INTEWATIONAL REVIEW OF CYTOLOGY. VOL. 76

Highly Repeated Sequences in Mammalian Genomes

               MAXINE F. SINGER
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health,
              Bethesda, Maryland

I. Introduction . . . . . . . . . . . . . . .
11. Perspectives on Methods . . . . . . . . . .
III. Satellites . . . . . . . . . . . . . . . .

A. Changing Concepts . . . . . . . . . . .
B. Rodents . . . . . . . . . . . . . . .
C. Bovine . . . . . . . . . . . . . . .
D. Primates . . . . . . . . . . . . . . .
E. Marsupials . . . . . . . . . . . . . .
F. Carnivora . . . . . . . . . . . . . .
G. Comparisons between Satellite Sequences . . .
H. Chromosomal Location . . . . . . . . .
I. The Question of Satellite Function . . . . .
J. Amplfication of Satellites . . . . . . . .

IV. Interspersed Repeated Sequences . . . . . . .
A. Emerging Concepts . . . . . . . . . . .
B. Short Interspersed Repeated Sequences (SINES)
C. Long Interspersed Repeated Sequences (LINES)
  D. Amplification and Dispersion . . . . . . .
V. Concluding Remarks . . . . . . . . . . . .
  References . . . . . . . . . . . . . . . . .
  Note Added in Proof . . . . . . . . . . . .

..... 67
..... 68
..... 71
..... 71
..... 13
..... 77
..... 80
..... 86
..... 86
..... 86
..... 81
..... 88
..... 89
..... 91
..... 91
..... 92
..... 102
..... 104
. . . . . 105
_.._.. 106
...... 112

I. Introduction

The discovery of restriction endonucleases and the development of molecular
cloning and DNA sequencing techniques have revolutionized the study of the
structure of complex genomes. The unexpected insights generated by these new
techniques have created profound changes in our understanding of the way genet-
ic information is organized and expressed. Consequently, many new concepts
regarding the highly repeated DNA sequences in eukaryotic genomes are rapidly
developing. Perhaps the only generalization that can safely be made at this time
is that both the repeated sequences and their genomic organization are much
more complex than previously suspected. This article attempts to summarize the
emerging picture regarding the structure and organization of mammalian highly
repeated sequences at the molecular level. Both the tandemly repeated sequences

61

Copyright @ 1982 hy Academic Press. Inc.
All rights of reproduction in any form reserved.
            ISBN 0-12-364476-3

68 MAXINE F. SINGER

widely termed "satellites" and those segments that are interspersed among other
genomic DNA sequences will be described. As a general rule of thumb, any
sequence repeated more than 101 times is included. I have chosen to concentrate
on mammals because of my own interests and because other recent reviews
emphasize plants (Bedbrook and Gerlach, 1980; Havell, 1980) and invertebrates
including crabs (Christie and Skinner, 1980), Drosophila (Appels and Peacock,
1978; John and Miklos, 1979; Brutlag, 1980; Hilliker et aE., 1980; Spradling and
Rubin, 1981), and sea urchins (Moore et al., 1980; Posakony et al., 1981). The
available information is for the most part structural. Unfortunately, the function,
if any, of most of these sequences remains an enigma.
Most articles covered here were published prior to the end of July 1981 al-
though I had, through the courtesy of colleagues, preprints of other papers. My
own interests dictated selection of the material. The interesting matter of the
organization of highly repeated sequences within chromatin is discussed exten-
sively in several recent reviews (Zachau and Igo-Kemenes, 1981; Kornberg,
1981; Igo-Kemenes et al., 1982) and is not included here. Repeated sequences
that are known to be genes (e.g., histone and ribosomal RNA genes) have also
been reviewed recently (Long and Dawid, 1980). There are available several
summaries of current thinking about the evolution of highly repeated sequences
(Brutlag, 1980; Bostock, 1980; Dover, 1981). My own bias is that while relevant
structural information is rapidly accumulating, we are still largely ignorant of
ovemding principles. In particular, with the question of function unresolved,
discussions of evolution are perforce carried out in the absence of any sense of
selective pressure, a critical defect.
Throughout this article the words ``repeated ' ' and ` `reiterated ' ' are used inter-
changeably. Neither one implies that the many copies of a given family of
sequences are identical. Unless demonstrated otherwise, all "repeats " are as-
sumed to be members of a set of closely similar but somewhat variant DNA
segments; "closely similar" means that they hybridize to one another under
stringent conditions (0.45 M NaC1, greater than 65C). The term "consensus
sequence" will be used to describe a nucleotide sequence representing the most
abundant nucleotide at each position in the repeat units comprising a set.

11. Perspectives on Methods

In the past, two methods dominated analysis of repeated DNA sequences:
measurement of DNA renaturation kinetics (Britten et al., 1974) and isopycnic
centrifugation in gradients of CsCl and CsSO, (Szybalski, 1968). Gross physical
separation of highly repeated, middle repeated, and unique sequences was
achieved by taking advantage of (1) the dependence of renaturation rate on` the
concentration of a sequence, (2) the ability of hydroxyapatite to separate single-

REPEATED SEQUENCES IN MAMMALIAN GENOMES

69

and double-sfranded DNA, and (3) the separation of some satellite sequences
from bulk DNA by virtue of a unique density. Together, these methods demon-
strated that (1) eukaryote genomes can be divided up into classes of DNA
sequences according to the reiteration frequency (Britten and Kohne, 1968), (2)
many very highly repeated sequences are in long tandem arrays (satellites), and
(3) some repetitive sequences are dispersed throughout major portions of
genomes amid either other repeated sequences or sequences present only once
per genome, that is, "unique" sequences.
Even before molecular cloning came into wide use, it was apparent that these
useful characterizations masked much greater complexity. For example, the mix-
ture of sequences in the ``middle repeated" category include; segments repeated
anywhere from two times to tens or hundreds of thousands of times. Some of
these sequences proved to be functional genes+ither identical multicopy genes
such as those for ribosomal RNA and histones, or closely related genes encoding
similar but distinctive gene products such as the families of actin, P-globin, or
immunoglobulin genes (see review by Long and Dawid, 1980). Furthermore,
because divergence among the members of a set of repeated sequences decreases
the rate at which they reassociate, middle repeated sequences can appear in the
single copy class and the copy number of highly repeated sequences is easily
underestimated. Similarly, although many organisms show one or more discrete
satellite DNA fractions apart from the main band upon isopycnic centrifugation,
others yield no such fractions. Centrifugation in the presence of heavy metals or
various antibiotics or dyes, tease "cryptic satellites" out of the DNA of many
organisms, but others yield no satellite even though renaturation kinetics shows
the presence of highly repeated sequences.
New methods have now begun to unravel some of the complexities and it is the
application of these methods that is the basis for the observations summarized in
this article. Restriction endonucleases cleave DNA molecules after recognition of
specific short nucleotide sequences. The recognition and cleavage sites of some
of the enzymes referred to in this article are shown in Table I. Complete current
lists of known restriction endonucleases are available (Roberts, 1980). Digestion
of DNA with a specific enzyme reproducibly divides it up into a set of fragments
whose sizes depend on the spacing between recognition sites. The mixture of
fragments can be conveniently separated according to size by electrophoresis on
semisolid supports such as agarose or polyacrylamide (Southern, 1979). When
the gels are stained to mark DNA the mixture appears as a continuous smear of
fragments of all possible sizes. However sequences that are repeated sufficiently
to represent at least 0.5% of the genome often appear as distinct bands against the
background smear. The entire collection of fragments on the gel can be trans-
ferred without disturbing their distribution by blotting after denaturation onto
sheets of nitrocellulose (Southern, 1975b, 1979) or diazotized paper (Alwine et
al., 1979). Thereafter, the sheets can be incubated in the presence of radioactive

70

MAXINE F. SINGER

              TABLE I
RECOGNITION AND CLEAVAGE SITES OF SOME RESTRICTION
             ENDONUCLEASE^

Enzyme Sites

Sau96I

AvuU

TuqI

Sau3A
MboI

5`-G`G N C C -

-C C N GIG -5'

A

5'-G`G (T)C C -

    A

-A G C` T -5`

-C C (T)G'G -5'

5`-T' C G A -

5'-'GAT C-

-C T A G'-5'

EcoRI 5`-G'A A T T C-

-C T T AA'G-5`

AluI 5'-A G'C T -

-T C'G A -5'

Hindu1 5`-A`A G C T T-

-T T C G A`A-5'

BumHI 5'-G'G A T C C-

-C C T AG'G-5`

a Taken from Roberts (1980) where a complete list can be found. The
indicates the site of cleavage within the recognition site. Enzymes with

identical specificity (Suu3A and MboI) are called isoschizomers.

DNA or RNA fragments to permit hybridization of the probe with homologous
sequences on the sheet. Exposure of the sheet to X-ray film provides an au-
toradiogram on which a darkened band reveals the DNA fragments homologous
to the probe. These techniques are sufficiently sensitive to reveal even unique
DNA sequences amid fragments generated from an entire mammalian genome.
Molecular cloning, usually in E. coli host vector systems, provides ways to
purify and amplify DNA segments generated after restriction endonuclease diges-
tion or after random generation of genomic fragments by shearing or nonspecific
nuclease digestion (Morrow, 1979). Hybridization techniques with radioactive
probes permit identification of the clones of interest and primary nucleotide
sequence is readily determined (Maxam and Gilbert, 1980; A. J. H. Smith,
1980). In this way, single molecular species corresponding to the individual
members of a repeated set are available as are segments corresponding to a
tandem array or a dispersed repeated sequence enclosed within its natural
neighbors. One very important consequence of these methods is the availability
of pure DNA segments for use as probes in the analysis of genome orga?ization
by the blotting procedures described above. Uncloned probes are likely to be

REPEATED SEQUENCES IN MAMMALIAN GENOMES

71

contaminated by extraneous DNA sequences. Because of the very high sensitiv-
ity of the blotting methods even minor contaminants lead to misleading data. A
word of caution: molecular cloning is not without problems. Many investigators
have noted that deletions often occur when tandemly repeated arrays are cloned
and amplified in E. coli (e.g., Brutlag et al., 1977; Carlson and Brutlag, 1977;
Sadler et al., 1980; Sakano et al., 1980).

111. Satellites

A. CHANGING CONCEPTS

The characteristic organizational feature of satellites and cryptic satellites is the
tandem repetition of a unit DNA sequence. In accordance with a previous sugges-
tion (Pech et al., 1979b), the term satellite will be used to describe such DNA
regions regardless of whether or not they are separable as classical satellites by
isopycnic centrifugation. This usage, while not completely accurate, conforms
with widespread practice. Satellites comprise anywhere from a few percent
(human) to over 50% (Kangaroo rat) of mammalian genomes. Common char-
acteristics of satellites include (1) association with heterochromatin, (2) lack of
measurable transcription (but see below), (3) replication late in S-phase, and
(4) underreplication in polytene chromosomes (see Brutlag, 1980, for review).
At one time it was believed that all satellites contain simple redundant repeats
of short oligonucleotide segments. However molecular analysis has demon-
strated that repeat units vary from a few to several thousand base pairs and that
enormous complexity resides within these sequences. Satellite arrays frequently
resist separation by isopycnic centrifugation and remain instead within the main
density fraction of genomic DNA. This is true even when some satellite is
separable by centrifugation; additional satellite may remain sequestered in the
main band. Furthermore, neither the punty nor the unique character of an
isopycnic satellite fraction can be assumed. Sequences present in one satellite
band may also occur in others and in main band. Also several nonhomologous
repeat units may reside in one density satellite fraction, either linked in one
molecule or on separate molecules.
Regardless of whether separated satellite or total DNA is used, both the repeat
unit and its tandem organization can be revealed by restiiction endonuclease
digestion. When a site for a particular restriction endonuclease occurs within a
typical repeat unit, digestion converts a tandem array to a set of DNA fragments
of repeat unit length (a type "A" digestion; Horz and Zachau, 1977). Partial
digests generate "ladders" of fragments that are demonstrable upon elec-
trophoresis. The ``ladder'' fragments are integral multiples of the basic repeat
unit in length, and provide evidence for the tandem organization of the satellite.

72 MAXINE F. SINGER

After exhaustive digestion, a "ladder" of resistant multiples usually remains.
The resistant ladder arises because of sequence alteration at the canonical restric-
tion endonuclease site in occasional copies of the repeat unit. Such alterations
may be (1) randomly dispersed among the copies of the unit repeat, or (2)
clustered within neighboring repeats, or (3) occur at regular intervals along an
array. All three possibilities occur. The latter two nonrandom arrangements (and
combinations of the twb) define distinctive subarrays of a satellite and these are
here called satellite domains. Restriction endonuclease sites that are missing
from the typical unit repeat sequence may occur occasionally within an array
because of sequence variation and yield distinctive digestion patterns with a
preponderance of very long fragments (a type "B" digestion). Again the same 3
distributions occur and may define specific satellite domains. The absence of an
A-type site or the presence of a B-type site at a fixed frequency within an array of
repeats is seen as a long repeat length superimposed on a shorter canonical
reiterated unit.
The primary nucleotide sequence of a repeat unit can be determined using an
entire set of monomeric units generated by type A digestion or a molecularly
cloned member of the set. Unambiguous consensus sequences are frequently
obtained with uncloned sets, indicating that variant bases occur in fewer than
15% of the copies at any given position. The 15% limit reflects the limits of
detectability in the DNA sequencing procedures. In the typical case one restric-
tion endonuclease is used to generate monomer units; any repeat units lacking the
site appear in the higher ladder and are not represented in the consensus. Sub-
sequent steps in the preparation of fragments for sequencing often utilize
additional restriction sites in the repeat unit to generate subunit size sections. At
each such cleavage repeat units lacking the site in question will be discarded with
undigested material and will not be represented in the consensus. Thus, the
existence of subsets of a given monomer bias the sequence data on uncloned
fragments.
Some organisms have multiple distinguishable satellites. Sequence analysis
frequently shows that these may be related to one another in spite of different
densities and restriction endonuclease cleavage patterns or inability to cross
hybridize. Other organisms appear to have one predominant satellite. In these
cases, molecular analysis may show the presence of domains. It is possible that
domains and different satellites are organizationally equivalent, representing
localized amplifications of particular variants of a basic sequence. There is
growing evidence that different domains or distinguishable satellites may tend to
be localized to specific chromosomes. Both the domains of a relatively
homogeneous satellite and the several satellites in some organisms can all be seen
as the product of alternating cycles of mutation (including single base pair
changes, deletions, and insertions) and amplification and deletibn. This scheme,

REPEATED SEQUENCES IN MAMMALIAN GENOMES

73

P

1 Amplification

W~ I IFIIJ

I Divergence (transition, transversion, deletion, insertion)

Lcu(=III=yu P

1 Amplifkation

u, I =I I I., , -..rgr IX IX 19 I 111 w

I Deletion

- ' .' =I - 1 etc.

FIG. 1.

Schematic diagram describing the generation of related but divergent satellites, including

domains, by alternating cycles of mutation and amplification (after Southern, 1970).

first suggested by Southern (1970), is outlined in Fig. 1; it is consistent with the
data on various satellites, as reviewed below.

B. RODENTS

1. Mouse

The single density satellite of Mus musculus makes up 5 to 10% of the genome
(Kit, 1961) and is localized at centromeric heterochromatin in all mouse chromo-
somes except the Y chromosome (Pardue and Gall, 1970). Most (60-70%) of
isolated mouse satellite is digested to a set of 234-bp-long monomeric units by
Suu96I (Horz and Altenburger, 1981), EcoRII (Southern, 1975a; Horz and
Zachau, 1977), and AvuII (Dover, 1978). The set yields an unambiguous con-
sensus sequence (Horz and Altenburger, 1981; Manuelidis, 1981a) (Fig. 2a).
The asymmetric distribution of A and T residues on the two DNA strands of the
repeat unit may account for anomalous prior estlmates of both the size of the
repeat unit (by gel electrophoresis) and of the GC content (by density gradient
centrifugation or melting temperature determination). The 234-bp segment com-
prises four related internal tandem repeats-58, 60, 58 and 58 bp in length,
respectively. And each of these can be further divided into two related but variant
28- and 30-bp-long segments. Thus the 234-bp repeat encloses 8 shorter tandem
repeats. Horz and Altenburger (1981) deduced a common progenitor sequence

74

MAXINE F. SINGER

a

10 20

70

130

190 200 210

TTGAAAAATG ACGAAATCAC TAA+AACGT

      40
TCACGGAAAA

     100
GGTGGAAAAT

     160

ATGGAAAATG AGAAACATCC ACTTG GAC

230 1

220

GAAAAATGAG AAATGCACAC TGAA

10 20 30 40 50 60

b
EAATTcAcAG AGAAAcAGTG TTTCAGTTCG TTAAAACGTT GCTCTATCTT GAATAACAAG

                                       110 120

                                       170 180
CTTATTACAT SCGAATCCTA TTGGGAACCT AC AATTCA CCATGATACT TAGATTCC6T

TCCTCAAAAT GTTGCTCCAT ATTGAAAAGC AAACTCATAC AAGCAGGTCC CATTGGGAAC

            200 21 0 220 230 240
TCAC AATT CGCCTAGAAA TTTTGATTCC ATTCGTGAAA ATTTTTCTAT ATCCCGAACA

                                       290 300

                                       350 360
GTCCACTTAT TACTACTGCG GCCCACTGGG ATTCACCATG TTACTCAGAT

TCGGCTCACC AAATTTTGAT AAATCTTTAA AAGTACACAT ATTACAAGAG CAGGCTACTG

90 I loo

70 80

130 140 150 160

260 270

P I9O

250

31 0 320 330 340

     370
GGAACTAACT

1

FIG. 2. The consensus sequences of the major monomeric repeat units in (a) mouse satellite (Horz
and Altenburger, 1981) and (b) rat satellite 1 (Pech et al., 1979b). Brackets enclose the long internal
repeats and lines mark off the 8 shorter internal repeats in the mouse sequence. Only one DNA strand
is shown and it is 5' to 3' reading from left to right.

from the structure of the 8 repeats and suggested that the satellite may have been
constructed by alternating mutation and amplification starting with three similar
nonanucleotides: GAAAAATGA , GAAAAAACT , GAAAAACGT.
Several variants of the 234-bp consensus sequence have been identified. Thus,
approximately 5-10% of the 234-bp-long fragment set contains a single TuqI
cleavage site that is missing in the consensus sequence (Horz and Altenburger,
1981). Approximately 20% of the satellite is degraded to dimers (464 bp) after
exhaustive cleavage with either Suu961 or EcuRll (Horz and Zachau, 1977;
Southern, 1975a) and thus variation at these (overlapping) restriction sites (resi-
dues 1-8 in Fig. 2a) is frequent; the dimer yields a consensus sequence almost
identical to that of the monomer (Horz and Altenburger, 1981). Suu96I digestion
also yields small amounts of heretical fragments 0.25 X n X 234 bp in length (n
is an integer); these structures have not been studied in detail. There is extensive
methylation of C-G sequences in the satellite (Horz and Altenburger, 1981).
Further, some sequences that hybridize with mouse satellite pe found in DNA

REPEATED SEQUENCES IN MAMMALIAN GENOMES

75

from which satellite has been removed by density gradient centrifugation (Man-
uelidis, 1980; Stambrook, 1981).
Individual mouse chromosomes may carry distinct satellite domains. DNA
from a Chinese hamster/mouse hybrid cell line containing only the mouse X
chromosome in 75% of the cells was studied by restriction endonuclease diges-
tion and hybridization with 32P-labeled density gradient purified satellite (Brown
and Dover, 1980a). There is no cross-reaction with the Chinese hamster DNA.
The data indicate that the X-chromosome contains mouse satellite domains
whose basic organization is similar to the bulk of the satellite but that (1) contain
fewer Hinf, AluI, and EcoRI sites, and (2) lack heretical size fragments.
Sequences homologous to the M. musculus satellite have been detected in
other Mus species by hybridization with radioactive probes of M. musculus
satellite purified by density gradient centrifugation; the data require confirmation
with cloned probes. There was less in situ hybridization to M. booduga chromo-
somes than to those of M. musculus although the distribution was similar; only
one segment on the short arm of the X-chromosome in M. dunni showed signifi-
cant hybridization (Sen and Sharma, 1980). Hybridization to restriction endonuc-
lease fragments of total M. spretus DNA (Brown and Dover, 1980b) indicated
sequences homologous to M. musculus satellite at about 1% the level even
though M. spretus does not yield a classical density satellite. Almost all the
homologous material in M. spretus was degraded by AvaII, as is M. musculus
satellite. AluI, which degrades about 10% (type B) of M. musculus satellite,
digested only about 2-3% of the homologous sequences in M. spretus to sepa-
rable products suggesting different subsets of sequences in the two species.
Restriction endonuclease digests suggest the presence of related satellite se-
quences in two species of the field mouse genus Apodemus (Brown and Dover,
1979).

2. Rat

No substantial satellite, cryptic or otherwise, can be isolated from total R.
ruttus DNA by isopycnic centrifugation, although renaturation kinetics shows
that almost 10% of the rat genome comprises highly repeated sequences (Bonner
et al., 1973). Digestion of total rat or Novikoff hepatoma ascites cell DNA with
several restriction endonucleases yields ladders of DNA fragments of defined
chain length, indicative of the presence of tandem repeated sequences
(Philippsen et ul., 1974; Maio et al., 1977; Fuke and Busch, 1979; Lapeyre and
Becker, 1980; Sealy et al., 1981). Some distinct'differences in the pattern of
EcoRI bands observed with rat liver nuclear and Novikoff hepatoma DNAs have
been reported (Lapeyre and Becker, 1980). A predominant 370-bp-long HindIII
band is degraded by EcoRI to yield bands about 93 bp long, which is the size of
the smallest abundant band obtained by EcoRI digestion of total rat DNA. About
3% of the rat genome is accounted for by these fragments (Fuke et al., 1979;

76 MAXINE F. SINGER

Pech et al., 1979b) and the sequences may be concentrated in nucleolar DNA
(Fuke et al., 1979).
Approximately 1-3% of rat DNA is converted to fragments about 10 kbp in
length by digestion with Sau3A (type "B" digestion) while most of the rest of
the genome is simultaneously degraded to a large mixture of small fragments
whose average chain'length is 400 bp (Pech et al., 1979b). The 10-kbp fraction
was purified by preparative gel electrophoresis and named rat satellite I. Analysis
of the purified satellite I (Pech et al., 1979b) demonstrated a repeat length of
370 bp constructed of four related internal segments 93, 92, 93, and 92 bp in
length, respectively (Fig. 2b). Each of the internal segments contains an EcoRI
site (at residues 1-6, 94-99, 186-191, and 279-284 in Fig. 2b) and one contains
a Hind111 site (residues 58-63). The sequence data obtained with the set of
Hind111 monomers (Fig. 2b) revealed frequent variations at eight defined resi-
dues. These must represent alterations in at least 5-10% of the members of the
set. Thus a substantial number of the members diverge in specific nonrandom
ways. It is not known whether the different abundant variants are present in the
same or different copies of the sequence. Domains have not been characterized,
but they may occur since some portion of the sequence is left uncut by each of the
restriction endonucleases with otherwise regular sites. The CpG sequences in the
satellite are extensively methylated (Pech et al., 1979b). Independent base se-
quence determination on EcoRI fragment sets 92 and 93 bp in length, respec-
tively, and generated by cleavage of total rat DNA with EcoRI confirms the
relatedness of the EcoRI fragments to rat satellite I (Lapeyre et al., 1980; Sealy
et al., 1981).
As with R. rattus neither satellites nor cryptic satellites can be isolated from
R. norvegicus, R. sordidus, or R. villosissimus by isopycnic centrifugation. Also,
less than 5% of the three genomes are highly repeated segments (Miklos et al.,
1980). Analysis by restriction endonuclease digestion is consistent with the pres-
ence of very low amounts of satellite DNA in these three species.

3. Other Rodents

The oligomer 5'-TTAGGG-3' is repeated frequently in a major satellite, Hs,
of Dipodomys ordii, the kangaroo rat (Fry and Salser, 1977) and in guinea pig
a-satellite (or satellite I) (Southern, 1970). The same oligomer probably occurs
frequently in Thomomys bottae, the pocket gopher, Ammospermophilus
leucurus, the antelope ground squirrel, and other species in the genus Dipodomys
(Fry and Salser, 1977; Mazrimas and Hatch, 1977). Analysis of the total repeti-
tive DNA of the guinea pig, Cavia porcellus, was made by isolating the fraction
reassociating at a Cot of about 7 X   and removing remaining single strands
with SI nuclease (Hubbell et al., 1979). The data suggested that about 21% of
the genome of C. porcelhs is highly repeated while earlier estimates, based on
density gradient isolation of satellites indicated about 10%. It is possible that the

REPEATED SEQUENCES IN MAMMALIAN GENOMES

77

rapidly annealing fraction contains both satellites and interspersed repeated se-
quences as it is heterogeneous. The three C. porcellus satellites that are separable
by isopycnic cehrifugation were used to prepare 3H-labeled cRNAs and the
probes were hybridized in situ, to C. porcellus chromosomes (Duhamel-
Maestracci et al., 1979). All hybridization was to centromeric regions and some
distributional specificity of the 3 satellites was observed; the Y chromosome
hybridized to none of the three probes. Confirmation of the data with cloned
probes is required.

C. BOVINE

The eight different satellites that have been distinguished in calf thymus DNA
(Macaya et al., 1978; Kopecka et al., 1978) are listed in Fig. 3 along with their
buoyant densities in CsCI, their relative abundance in the genome, and, where
known, a schematic diagram of the organization of the repeat units. The purifica-
tion of these satellites depends on sophisticated use of differential density grad-
ient centrifugation in CkSO, in the presence of various additives (Macaya et al.,
1978; Streeck et al., 1979). Together, the eight comprise over 23% of the
genome although analysis of the kinetics of renaturation of calf thymus DNA
suggested that less than 5% was in very rapidly renaturing components. This
dramatic example emphasizes that satellite and ``rapidly renaturing" are not
synonymous. Recent analysis of bovine satellites on the molecular level illus-

BUOYANT %

NAME DENSITY GENOME ORGANIZATION

ABCD

111 1.706 4.2 2350

1200 247 650 251

1.709 4.6

1.711a 1.7 . ... . 1413

1.711b 7.1 0 :.:.. 2600

1.715 5.1 -1400

1.720a 0.1

NC I D

E I' E

     E

550 611 252

-

1.720b     0.1 46

1.723    0.5 -700

    Bovine satellites and their structural organization where known. See the text for refer-
ences. The earlier "names" of the bovine satellites are given in the first column; the identity of II is
uncertain. Closely related segments repeated in more than one satellite are indicated graphically and
by letter. The numbers to the right are the sizes, in base pairs, of the overall repeating units. The sizes
of different segments are indicated below. The drawings are not to scale.

FIG. 3.

78 MAXINE F. SINGER

trates how distinctions based on satellite density or even restriction endonuclease
digestion can obscure striking similarities. Also, these studies show that the size
of repeat units determined by restriction endonuclease digestion is not a reliable
indicator of underlying structure. In one case, the 1.706 gm/cm3 bovine satellite,
what initially seemed a complex satellite with a repeat unit of 2350 bp, has turned
out to have true repeat unit about 0.01 times that size! And at least 5 of the 8
satellites are related in part to one another.
Digestion of purified 1.720b satellite with several different restriction en-
donucleases yields a series of fragment sets that are multiples of 46 bp in length
[Streeck and Zachau, 1978 (note that the 1.720b satellite is eKoneously termed
1.723 in that paper), Poschl and Streeck, 19801. The set of 46-bp fragments
produced by AM gave an unambiguous consensus sequence (Fig. 4) and re-
vealed an underlying periodicity of two related 23-bp repeats. Individual copies
of the repeat units diverge from the consensus sequence. Whether or not particu-
lar divergent farnily members are collected in domains is not clear. The sequence
contains several CG dinucleotides and these are probably frequently methylated.

Although quite distinct by density and restriction endonuclease patterns, the

I I I t

1.72Ob 5 -TATCAGGCAGATGAGCGGfCGCGCGGCTCAGCTGGCGAG

1.706A 5 I 'GATCACGTGACTGATCATGCACT -

1.706C S1-GATCACGTGGCTGATCATGT

1.7068 5 I -MTCATGCAGCTCAGCAGGCA$

1.706D 5*-MTCAAGCAGGTCAGCAGGCAAT

    5 I - GATCACGTGACTGA!$A%CACT

        5' -AATCACGCAGCTCAGCAGGCAAT

1.7 1 la A f C

1.7 1 la BID

r

1.706A(L) GATCACGTGACT

L706A( 2) GATCATGC ACT

FIG. 4.  Sequence relations among the bovine satellites (see Fig. 3). The 46-bp consensus unit of
the 1.72Ob satellite is from Poschl and Streeck (1980); the 23-bp consensus units of 1.706A, B, C,
and D are from Pech et al. (1979a); the 23-bp consensus units of 1.711a A/C and B/D are from
Streeck (1981). Only one DNA strand is shown in each case. The data are somewhat simplified and
the reader is referred to the original papers for details.

REPEATED SEQUENCES IN MAMMALIAN GENOMES

79

sequence of the 1.706 satellite (111) is related to that of the 1.720b satellite. The
overall repeating unit of the 1.706 satellite (Streeck and Zachau, 1978; Streeck et
al., 1979; Pech eta!., 1979a) contains four distinct regions totaling 2350 bp. The
distribution of restriction endonuclease sites and direct nucleotide sequencing of
both cloned and uncloned fragments revealed that the four regions comprise two
pairs of closely related alternating segments (A and C, B and D). The two kinds
of segments (NC, B/D) are themselves distantly related. The underlying unity is
the tandem repetition of a 23-bp unit that is related to the repeat unit of the
1.720b satellite (Fig. 4). Furthermore, the 23-bp repeat unit of 1.706 A, for
example, itself looks like a tandem repeat of a unit half its size (see Fig. 4).
The 1.711a satellite (Streeck, 1981) is also related to 1.720b and 1.706; an
unambiguous consensus sequence was obtained with an uncloned fragment set.
The long-range repeat length of 1.711a is 1413 bp and can be divided into 3
regions. Two are marked A/C and D in Fig. 3 to reflect their marked similarity
to the corresponding segments in the 1.706 satellite, and the third region, which
is unrelated to A/C or D and is not internally repetitive, is marked I. The I
region has several features reminiscent of transposable elements (reviewed by
Calos and Miller, 1980) including terminal direct and inverted repeats (Streeck,
198 1). Indeed, the 1.7 1 1 a repeat unit can be viewed as one portion of the 1.706
unit interrupted by the I region. Furthermore, I contains open reading frames
suitable for translation into protein and a segment similar to the TATAAATA
box that is one component of eukaryotic promotors.
More than 95% of the 1.715 satellite from either calf thymus or bovine kidney
cells in tissue culture is degraded by EcoRI and by Sal1 to a fragment set about
1400 base pairs in length (Botchan, 1974; Roizes, 1974, 1976; Philippsen et al.,
1974; Lipchitz and Axel, 1976; Gaillard et al., 1981). Analysis of the 1400-bp
monomer set with other restriction endonucleases established that the bulk of the
set shares similar sequences but that variant forms exist (Roizes, 1976; Roizes
et al., 1980). At least four distinguishable domains may occur. Superimposed
on this nonrandom division into domains other variations appear to reflect ran-
dom base pair changes leading fortuitously to altered (gain or loss) restriction
sites (Roizes et al., 1980). The sequence of the 1.715 satellite monomer does
not contain tandemly reiterated internal repeats like the 23 bp regularity found in
satellites 1.720 and 1.71 la but does contain multiple repeats of short segments
of the consensus sequences of those satellites (Roizes et al., 1980; Gaillard et
al., 1981; A. Plucienniczak, J. Skowronski, and J. Jaworski, personal com-
munication). Comparison of restriction sites in the cloned and sequenced mono-
mer with those in the bulk satellite suggests extensive' methylation (Roizes et
al., 1980; Kaput and Sneider, 1979; Gaillard et al., 1981). The extent of methy-
lation appears to differ markedly in sperm and thymus DNA (Sturm and Taylor,
1981).

80 MAXINE F. SINGER

The 1.711b satellite is similar to 1.715 except that the 1400-bp repeat unit is
interrupted about 60% of the time by a 1200-bp-long insertion (I` on Fig. 3). The
arrangement of 1.7 1 lb repeat units with and without the I' element has no known
pattern. I' shares sequence homologies with the I element in 1.711a (Streeck,
1981). The I' region of 1.71 lb has inverted terminal repeated sequences and is
flanked by duplications of the 6 base pairs at the point of interruption of the
1400-bp repeats. In contrast, the I region in 1.7 1 la is not surrounded by direct
repeats of the target sequence.

D. PRIMATES

1. a-Satellites

At least one satellite in every primate that has been investigated is part of a
kinship of sequences referred to as a-satellite (or alphoid) after the prototypical
African green monkey (AGM) (Cercopithecus aethiops) satellite (Maio, 197 1;
Kurnit and Maio, 1974). The AGM a-satellite represents about 20% of the
genome (Maio, 1971; Fittler, 1977; Singer, 1979), hybridizes to centromeres
(Kurnit and Maio, 1973, 1974; Segal et al., 1976), and comprises a substantial
amount of nucleolar DNA (Kurnit and Maio, 1973). Both in situ hybridization to
chromosomes (Segal et al., 1976) and kinetic analysis of interspersion frequency
(Singer, 1979) suggested that some copies of the satellite might be dispersed into
other parts of the genome. However this suggestion has not been confirmed on a
molecular basis and the kinetic data may reflect the interspersion of Alu se-
quences (see below) rather than a-satellite. Restriction endonuclease analysis
and hybridization experiments suggest that related but nonidentical a-satellites
are present in Gorilla gorilla, Pan troglodytes, Cercocebus aterrimus, Macaca
mulatta, Mandrillus sphinx, Papio cynocephalus, Cercopithecus pygerythrus,
Colobus badius, and Cebus capuchinus (Donehower and Gillespie, 1979; Singer
and Donehower, 1979; Gillespie, 1977; Musich et al., 1980; Maio et al.,
198 la). Typically, digestion of total or satellite DNA with one or another restric-
tion endonuclease yields a ladder of fragments that are integral multiples of
approximately 170 bp in length.

Sequence analysis confirms the presence of related a-satellites in AGM
(Rosenberg et al., 1978), baboon (Papio papio) (Donehower et al., 1980),
bonnet monkey (Macaca radiata) (Rubin et al., 1980a), and human (Homo
sapiens) (Manuelidis and Wu, 1978; Wu and Manuelidis, 1980) DNA (Fig. 5).
The length of the basic repeat unit is 172 bp in the AGM and close to twice that
in the three other species. The 2n units are composed of two different variations of
the basic repeat unit (an a-b-a-b-a-b type structure, see legend to Fig. 5). No regu-
lar internal periodicity has been discerned although large portions of the sequences
are alternating blocks of purines and pyrimidines (Rosenberg et al., 1978) and

REPEATED SEQUENCES IN MAMMALIAN GENOMES

81

1

2.

Zb

k

3b

4.

bb

1

2.

2b

3.

3b

6.

4b

1

2.

Zb

3.

3b

bm

bb

FIG. 5.

t

MGCAMTATCTTCCGTTCAMACTGGAMGA

MGGAMTATCCTCCGATMCAMGAGAMGA

MGGAMTATCCTCAGATGAMTCTGGAMGA

MGGAMTATCCTCAGATGAMTCTGGAMGA

MGGAMTATCCTCCGATMCAMGAGAMGA

TAGGAMTATCTTCCTATAGMACTAGACAGA

ACCGGAlT TCTTCATATTATG CTAGACAGA

     Sequence relations among primate a-satellites. (1) African green monkey (Rosenberg et
al., 1978); consensus sequence of uncloned HindIII monomer (172 bp). The sequence. is displayed
from one HindIll site to the next. (2) Baboon (Donehower et al., 1980); consensus sequence of the
dimeric uncloned BamHI monomer (343 bp). The two portions should be read a then b. The
sequences are aligned with the African green monkey sequence. The BanHI cleavage site is between
residues 115/116 in a. (3) Bonnet monkey (Rubin ef al., 1980); consensus sequence of the dimeric
uncloned HaeIII monomer (343 bp). The two portions should be read a then b. The HaeIII cleavage
site is between residues 137-138 in b. (4) Human (Wu and Manuelidis, 1980); consensus sequence
of the dimeric uncloned EcoN monomer (340 bp). The two segmwts should be read a then b. The

  cleavage site is between residues 2/3 in a. In each case only a single strand is shown; it reads
5' to 3', left to right.

there is a high frequency of 5'-GAAA and 5'-CTTT on the displayed strands
(Donehower et al., 1980; Rubin et al., 1980a).
The sequences of cloned monomers of the AGM satellite indicate that the
consensus sequence shown in Fig. 5 reflects a mixture of many versions that

82 MAXINE F. SINGER

differ from one another at a few positions (Rosenberg et al., 1978; Graf et al.,
1979; Graf, 1979; Thayer et al., 1981). No cloned unit has a sequence identical
to that of the consensus sequence. The same situation may exist in the a-satellites
of other species since ``ladder'' patterns are always observed upon exhaustive
type "A" digestion (Rubin et al., 1980a; Wu and Manuelidis, 1980; Donehower
et al., 1980; Musich et al., 1980; Maio et al., 1981a). At least some variants of
the AGM sequence are yithin satellite domains. For example, as many as 10% of
the monomer units contain an EcoRI cleavage site between residues 31 and 32
[inspection of the consensus sequence (Fig. 5) shows that a single base pair
change at residue 31 (T+ G) in the consensus sequence yields an EcoRI site].
Digestion with EcoRI yields a typical ladder pattern indicating a clustering of
units with EcoRI sites (Fittler, 1977). Two long a-satellite segments containing
frequent EcoRI cleavage sites were recently isolated by molecular cloning
(McCutchan et al., 1982). Furthermore, essentially all the a-satellite in a single
AGM chromosome isolated within a mouse-monkey somatic cell hybrid contains
cleavage sites for EcoRI; a large percentage of this a-satellite also has cleavage
sites for HaeIII (T.N.H. Lee and M. F. Singer, unpublished) although this site is
present in less than 3% of total a-satellite (Fittler, 1977; Rosenberg et al., 1978;
Graf et al., 1979; Thayer et al., 1981). Heretical size a-satellite fragments are
generated in small amounts by HindIII digestion (Fittler, 1977; McCutchan et
al., 1982). Some tandem arrays of AGM a-satellite are interrupted (McCutchBn
et al., 1982) by dispersed repeated sequences such as Ah-SINE or Kpn-LINE
(see below) family members (Grimaldi et al., 1981; Grimaldi and Singer, 1982).
The frequency at which heretical repeat units and interruptions occur is not
known.

2. Classical Primate Satellites

By classical, I refer to fractions separable by density gradient centrifugation,
cryptic or not. In the African green monkey the overwhelming mass ofthe major
classical satellite is a-satellite; three or four additional classical satellites have
been noted but not characterized (Kurnit and Maio, 1974; Fittler, 1977).
The classical human satellites present a complex picture compounded by the
fact that similarly named preparations are not necessarily identical from labora-
tory to laboratory (Macaya et al., 1977; Manuelidis, 1978; Miklos and John,
1979; Mitchell et ul., 1979). Macaya and co-authors (1977) and Miklos and John
(1979) valiantly summarized the confusions. Recently some clarity has begun to
emerge, but evaluation of the literature remains difficult. In particular, data on
chromosomal location obtained by in situ hybridization with different satellites
are problematic because of the cross-hybridization of the fractions (see below)
and the use of uncloned probes; it will not be summarized here. The story of the
complex interrelations among the classical calf satellites (see ?hove) perhaps
hints at what we may, in the long run, expect to learn. Table I1 summarizes one

REPEATED SEQUENCES IN MAMMALIAN GENOMES

83

      TABLE I1
CLASSICAL HUMAN SATELLITES"

Estimated percentage
  of genome

I
I1
III
IV

1.687
1.693
1.697
1.700

0.2-0.5
1-2
1-3
0.5-2

aData summarized from Macaya et al. (1977) and Mitchell et al.

(1979).

overview of the properties of the major classical human satellites I, 11, III, and IV
(Corneo et al., 1968, 1970, 1971; Macaya et al., 1977; Mitchell et al., 1979).
Together they represent only 2-5% of the genome although fractionation of
human DNA on the basis of renaturation kinetics suggests that this may underes-
timate the total (Marx et al., 1976; Schmid and Deininger, 1975). The percent-
ages of G- C base pairs estimated from the densities and melting temperatures are
at variance with one another (Macaya et al., 1977; Mitchell et al., 1979). Fur-
thermore, Mitchell et al. (1979) have presented data indicating that the density
fractions contain related sequences. Trace amounts of 32P-labeled I11 or IV reas-
sociate with identical kinetics in the presence of an excess of unlabeled 111 and
the melting curves of the resulting labeled duplexes are indistinguishable. Satel-
lites I and 11 each share common sequences with 111 (and thus presumably IV);
32P-labeled I1 hybridizes with 111, as does 32P-labeled I. The 111-like sequences in
I and I1 are probably not the same. It is not surprising then that all the satellites
hybridized in situ to the same regions of the same human chromosomes. The
classical satellites do not tell the whole story either; substantial satellite sequence
may be buried in the main density band of DNA (Corneo et al., 1980).
Of all the classical human satellites, 111 has been best studied (assuming that
what is called I11 is the same in each instance). It contains at least four different
types of sequence as defined by restriction endonuclease products and the ability
to cross hybridize. The extent to which the different sequence types are cova-
lently linked together is not known.
One portion of satellite I11 is degraded to a ladder of bands n X 170 base pairs
in length by restriction endonuclease HaeIII (Manuelidis, 1976; Bostock et al.,
1978; Mitchell et al., 1979, 1981). Similar bands y-e produced from satellite I1
(Mitchell et al., 1979). The repeat length of these units is immediately reminis-
cent of a-satellite type sequences and they may indeed be related. The uncloned
340-bp a fragment set (see Fig. 5) isolated from total human DNA after cleavage
with EcuRI hybridizes with HaeIII ladders produced by digestion of total

84 MAXINE F. SINGER

genomic human, chimpanzee, gorilla, and simiang DNA (Manuelidis, 1978;
Maio et al., 1981a). EcoFU digestion of satellite I11 itself yields a 340-bp frag-
ment (Mitchell et al., 1979). Confirmation of the relation between the EcoRl
dimer and sequences in the Hue111 ladder by hybridization with cloned probes is
required. Using uncloned total satellite I11 as a probe and rodent human somatic
cell hybrids that contained a limited number of identified human chromosomes
Beauchamp et aZ. (1979) found that certain size classes of Hue111 generated
fragments typical of total satellite are missing on specific chromosomes.
A second type of sequence included in satellite I11 is constructed of imperfect
tandem repeats of the sequence 5'-TTCCA-3' (see sequence 2 and 3, Fig. 6)
(Deininger et al., 1981); one of these is localized to the Y-chromosome (see
below). A third type contains interrupted imperfect repeats of 5'-TTCCA-3'; at
least two quite different versions of this type have been characterized within
cloned segments (see sequences 1 and 4, Fig. 6) (Cooke and Hindley, 1979;
Deininger et aZ., 1981). One of them is localized mainly to chromosome 1
(Cooke and Hindley, 1979). Since all these different cloned segments were
chosen essentially at random, a very large number of different versions of the
5'-TTCCA-3' sequence may occur, each in long tandem arrays that constitute
domains. Satellite I1 may contain related sequences (Manuelidis, 1978; Mitchell
et al., 1979). Inspection of the a-satellite sequences in Fig. 5 reveals a marked
frequency of sequences like 5`-TTCC-3` or 5'-GGAA-3' (equivalent to 5'-
TTCC-3` on the other strand). This may bespeak a relation between the satellite
I11 sequences and a-satellite (Deininger et al., 1981).

A fourth class of sequences included in satellite I11 occurs only in male DNA
and thus resides on the Y-chromosome. Upon digestion with HaeIII, EcoRI, or
EcoRII, male DNA yields a 3.4-kbp fragment set that is not found in female
DNA (Cooke and McKay, 1978; Cooke and Hindley, 1979; Bostock et al.,
1978; Kunkel et al., 1979). This fragment set may account for 40% of the entire
Y-chromosome. Experiments with both cloned and uncloned 3.4-kbp fragments
show that at least 50% of the sequences within the 3.4 kbp set hybridize with
satellite I11 segments that are also abundant in female DNA. The remainder are
male specific. Both types of sequences may be linked together in the members of
the set (Kunkel et al., 1979). This suggests an interspersion of Y-specific and
Y-nonspecific sequence elements in a domain with a 3.4-kbp repeat length. The
Y-specific domain is located on the long arm of the Y-chromosome (Kunkel et
al., 1977; Bostock et al., 1978). Other domains that include the Y-nonspecific
portion of the Y-domains but not within a 3 .Ckbp repeat unit are represented in
different amounts and different arrangements (domains) in various autosomes
(Cooke and McKay, 1978). The properties of two cloned segments picked at
random from a population of repeated sequences confirm the ,earlier analyses
(Deininger et al., 1981). One of the two hybridizes only to male DNA (sequence
2, Fig. 6) while the other hybridizes both to the male 3.4-kbp fragment set and to

REPEATED SEQUENCES IN MAMMALIAN GENOMES

85

1. Located on chromosome 1

I I I 1 1 I I t

5' MTTCATTTGMGACMTTCCATT~TACCAATTGATGATGGTTA~GATTCCATTTGATGATGATTACATTCCAT

  TTCATCATMTTCCATTCGATTCCA~CGATTCCATTC............................

  ......... CGMTGMTGAGTCCATCCATTTCAATTTCATGATMTTCCATTCG~CMTTCGATGGTG~CCATTC

  GATT.....................................TTCATTCGATTCATTTGATGATGATTCATGCGCGATTCA

  TTACATGA~ACCCCTTTCA~CCATT~T~A~ATTCCATTC~TCCAT...................3'

2. Located on Y chromaon

1 I 1 1 I t I 1

5' TTMTTCCATTCCATTCCATTCCATTCCATTCCATTCCATTCCATTCCA~CC~CCATTCCATTCGTG~GATTCCAT

  TCCATTCCATTCCA~CCATTC~TCCATTACATTCCACTCG~GATTCCATTC~CCATTCCMTCCATTCCAT

TCCATTCC 3'

3. Located on Y chromsomc and elsewhere

I I 1 I I I I 1

5' CATTCMGAMGITCCATECCCCATTCCCTTCGATTCCATTCCATTCGATTCCATTCTACTCGATTCCMTCTTGTC

CATTCC~~TTCCATTCCATTCCATTGCATTCCATTTC

  CATTCA 3'

4. Location unknown

1 1 I I I 1 I I

5' TTATTCCATTAGATTCCATTCGATGATGATTCCATTCGATTCCAGATGATT~ATT~A~CA~GATGATGATT

CCATTCGAGTCCACTCGATCCCATTCGAGTCCATTCATPCA

GTCCATTCGATGATTCCACTCGATTCCATTAGATGATTCCATT~AGTCCA~GATT~CCATTCGATTCCATTCGAT

  TCCT 3'

FIG. 6.  Related but variant domains in human satellite III. (1) From Cooke and Hindley (1979);

(2, 3, and 4) from Deininger et al. (1981).

female DNA (sequence 3, Fig. 6). Both are constructed of regular repeats of
5'-TTCCA-3' and variants thereof; the differing variations in the two cloned
segments must explain the specificity of hybridization. Note that a total of four
segments, all included in satellite III and all built from variations of 5'-TTCCA-
3', have been described (Cooke and Hindley, 1979; Deininger et al., 1981); their

86 MAXINE F. SINGER

structures are summarized on Fig. 6. In the absence of primary sequence data,
the different hybridization specificities of the fragments would have obscured
their similarities.
Little is known about satellite structure in other primates except that a-type
sequences are ubiquitous (see above) and that satellite sequences in anthropoid
apes are similar to one another (Deininger and Schmid, 1976b; Marx et al.,
1979; Mitchell et al., 1981).

E. MARSUPIALS

Macropus rufogriseus, the red necked wallaby, contains a satellite that can be
isolated on CsCl gradients containing actinomycin D. The bulk of the satellite is
degraded by BamHI to sets of fragments that are from 1 to 5 times 2.5 kilobase
pairs in length (Dunsmuir, 1976; Dennis et al., 1980) and the repetition fre-
quency is 5 X 105 in the genome. The 2500-bp repeat may be internally repeti-
tive with a periodicity of about 300 bp. Secondary digests with other enzymes of
both total satellite DNA and monomer units allow a division into distinct variant
subsets of the basic structure. The variant subsets are clustered in eight domains,
A through H, the characteristic structures of which are shown on Fig. 7. Within
each domain, occasional repeat units have altered sequences that result in ladders
of fragments with different enzymes. A model for the evolution of the satellite
(see arrows on Fig. 7) was constructed on the principle that a restriction site
common to the units in several domains was present in a common progenitor.

F. CARNIVORA

Centromeric C-banding is rare or nonexistent in members of the order Carniv-
ora and early attempts to demonstrate a density satellite in Felis catus (Pathak
and Wurster-Hill, 1977) failed. Matthews et al. (1980) have now reported that a
cryptic satellite is revealed by density gradient centrifugation in the presence of
netropsin. Analysis by reassociation kinetics after purification of the satellite by
repeated centrifugation indicated a Cot+ of 7 x lo4 mole/seconds/liter.

G. COMPARISONS BETWEEN SATELLITE SEQUENCES

Brutlag ( 1980) has recorded some statistically significant similarities between
short sequences in the complex satellites of various species including Drosophila
and several mammals. Thus residues 31 through 53 in the AGM a-satellite
consensus sequence (Fig. 5, #I) are homologous to a portion of the 1.706D
bovine satellite and residues 73 through 98 of the human sequence (Fig. 5, #4a) are
homologous to a portion of the rat consensus sequence (Fig. 2b). Mouse satellite
can be added to the list. Residues between 7 and 17 of the AGM consensus

REPEATED SEQUENCES IN MAMMALIAN GENOMES

87

1 Kb

u B (3.3)
/\

C (9.8) F (5.9)

+

uD(197) tG(5.9)

+ +

E (19.7) t H (17.7)

Tft9T
Barn Pst Hha XmaHind
HI I I 111

FIG. 7. Domains (A through H) within the red necked wallaby satellite (Dennis et al., 1980). The

numbers in parentheses represent the approximate percentage of total satellite in each domain.

(S'CTGAGAAACTG) are more than 87% homologous to multiple regions of
the mouse consensus and are themselves repeated in part in 3 other positions of
the AGM consensus. Further, the sequence is related to the progenitor nonanu-
cleotides proposed for the mouse satellite (see above). Also, there is a significant
similarity (93%) between residues 91 to 103 of the AGM sequence and 123 to
136 of the mouse. Unfortunately, statistical significance is neither necessary nor
sufficient for biological importance. Questions about common and functionally
significant satellite sequences in a range of species await new approaches.

H. CHROMOSOMAL LOCATION

Are specific satellites or domains located on particular chromosomes? This
important question remains largely unanswered except for the few instances
already mentioned. Yet it is an important question for speculating about satellite
evolution and is relevant to certain proposals about satellite function. Early
experiments that used radioactive classical satellite preparatiQns or RNA copies
thereof as probes for in situ hybridization to chromosomes are now suspect not
only because of possible contamination with other sequences but also because of
the cross-hybridization of related but distinct satellites. In some instances, exper-
iments with cloned and characterized probes should afford definitive data. But
where different domains cross-hybridize (e.g., in the African green monkey and

88 MAXINE F. SINGER

the wallaby) chromosome specificity will have to be determined with isolated
chromosomes. Somatic cell hybrids are a stable source of some individual
chromosomes and this approach is being used (as reviewed above). Furthermore,
direct methods for the isolation of specific chromosomes are being improved
(Davies et al., 1981).

I. THE QUESTION OF SATELLITE FUNCTION

There is noexperimental evidence regarding the function of mammalian satel-
lite DNA. Speculation about function presently rests on the universal (but not
necessarily exclusive) association of satellite DNA with heterochromatin (Brut-
lag, 1980; John and Miklos, 1979). However, heterochromatin function is itself
not understood at the molecular level and most of the relevant information is
limited to simpler eukaryotes that are amenable to either genetic or cytogenetic
analysis or both. Reviews of earlier experiments and speculations are avaifable
(Bostock, 1980; Miklos and John, 1979; Hilliker et al., 1980; Brutlag, 1980;
John and Miklos, 1979; Orgel and Crick, 1980; ooolittle and Sapienza, 1980).
The available data are most consistent with the idea that satellite functions in
germ line processes (Bostock, 1980). The following two experimental systems
may prove relevant to the question of satellite function.

1. Transcription

Transcription of satellite DNA occurs on specific loops in oocyte lampbrush
chromosomes of newts (Varley et al., 1980a,b; Diaz et al., 1981; Gall et al.,
1981). Within these genomes, the satellite occurs both in centromeric regions
and at the chromosomal regions corresponding to the loops. In Notophthalmw
viridescens, hundreds of 9-kbp-long clusters of histone genes are each embedded
within variable lengths of satellite sequence (estimated to be up to 100 kbp long)
at the loops. Transcription may initiate at promotor sites preceding the histone
genes and then proceed through a cluster continuing into the downstream satellite
sequences (Varley et al., 1980b; Diaz et al., 198 1). This is viewed as a failure of
normal transcription termination but whether that failure is functionally signifi-
cant remains to be learned.
There is one report that rat satellite sequences are transcribed in HTC rat tissue
culture cells (Sealy et al., 1981). This is of great interest, since earlier experi-
ments with less sensitive techniques showed no transcription in a variety of
species of organisms and cell lines.

2. Centromeric Function

The structure and function of yeast centromeres are now being investigated at
the molecular level (Clarke and Carbon, 1980a,b; Hsiao and Carbon, 1981;
Fitzgerald-Hayes et al., 1982; Clarke et al., 198 1). The ability to couple precise

REPEATED SEQUENCES IN MAMMALIAN GENOMES

89

information on DNA structure with detailed genetic analysis makes this an espe-
cially powerful system. Using recombinant DNA techniques small circular DNA
molecules have been constructed that contain selectable yeast genetic markers
(e.g., genes required for synthesis of a particular amino acid) and a yeast DNA
replicator sequence. Such plasmids replicate autonomously in yeast cells but are
mitotically unstable and are rapidly lost from the population in the absence of
dependency on the marker gene. The addition of a DNA segment spanning a
centromeric region imparts both meiotic and mitotic stability to the plasmid. It
then behaves like a true minichromosome, and becomes a stable component of
the cells even in the absence of the selective pressure. During crosses involving
two minichromosomes bearing identical centromeres but distinguishable marker
genes, the two can apparently pair, moving to opposite poles in the first meiotic
division (Clarke et al., 1981). The centromeres of yeast chromosomes 3 (Clarke
and Carbon, 1980a) and 11 (Fitzgerald-Hayes et al., 1982) have been charac-
terized. Yeast does not contain satellite sequences and there are no markedly
repetitious segments in the defined centromeric regions. The essential regions of
centromeres 3 and 11 are not more than 627 and 900 base pairs in length,
respectively, and do not cross-hybridize with one another. The centromere 3
sequence is not repeated elsewhere in the genome. Yet both contain long A-T-
rich regions flanked by short (14 bp or less) regions of homology. One of the
latter is reported to display limited sequence homology with the 1.688 gdcd
satellite DNA of Drosophila melanogaster (Clarke et al., 1981; Hsieh and
Brutlag, 1979a).

J. AMPLIFICATION OF SATELLITES

Most satellites are restricted to one or a closely related group of species and
thus many appear to be of relatively recent origin. This in turn suggests that
satellites change in evolution more than many other genomic regions. Compare,
for example, the very close similarities between the p-globin regions (Barrie et
al., 1981) and the Alu families (see below) of the primate genomes with their
strikingly different though related satellite domains. Large scale deletions and
amplifications were required to arrive at present day satellites. The similarities
between satellites of related species led to the "library" hypothesis regarding
satellite amplification and deletion (Salser et al., 1976; Fry and Salser, 1977); a
common library (or set) of related sequences is available within related species
and different members of the library are amplified to differing extents in the
several organisms. At present there is no reason to sissume a preexisting or
common library. It is equally probable that newly divergent sequences were and
are candidates for amplification (Gillespie et al., 1980).
Mammalian genomic sequences can be amplified. The organization of the
multiple globin genes (Fritsch et al., 1980, 1981) demonstrates this phenomenon

90 MAXINE F. SINGER

in evolutionary time. The amplification of certain eukaryotic genes also occurs in
experimental time. For example, clones of murine tissue culture cells containing
many copies of the gene for dihydrofolic acid reductase (Alt et al., 1978; Dol-
nick et al., 1979) or of the complex of genes responsible for synthesis of aspartyl
transcarbamylase (Wahl et al., 1979) are readily obtained under appropriate
selective pressure. In at least one instance, amplification of the mouse dihydrofo-
late reductase gene was accompanied by amplification of mouse satellite (Bos-
tock and Clark, 1980). Once amplified, extra copies of these genes are readily,
but not necessarily, lost when selective pressure is removed. Several mechanisms
for amplification of tandem repeats have been discussed (Tartof, 1975; Botchan
etal., 1978; Schimke etal., 1980; Smith, 1976; Kurnit, 1979; Baltimore, 1981).
Among these, unequal crossing-over (Smith, 1976) is a relatively simple
mechanism that is consistent with available data and has been demonstrated
experimentally with the tandemly repeated ribosomal RNA genes of yeast (Petes,
1980; Szostak and Wu, 1980). In brief, homologous crossing-over between
nonallelic repeat units in a tandem array (on sister chromatids or homologous
chromosomes or homologous regions on nonhomologous chromosomes) yields
an unequal number of repeat units in the products of recombination. Besides
resulting in reciprocal amplification and deletion, unequal crossing-over provides
for the maintenance of sequence homogeneity in tandem arrays (Smith, 1976). It
also predicts that repeat units near the ends of tandem arrays will be more
divergent than those near the center (Smith, 1976; Brutlag, 1980), as found with
the AGM satellite (McCutchan et al., 1982). Another mechanism for assuring
homogeneity, called gene conversion, is described in Section V,D. This
mechanism may be relevant to the fact that satellites on different chromosomes
are characteristic of the species, even though they may be distinct domains
(compare for example the a-a-a-a type structure of African green monkeys with
the a-b-a-b-a-b of baboons).

Amplified genes, besides being subject to deletion, can follow at least three
additional courses. First, they may be used essentially as such, as are the two
adult a-globin genes. Second, they may evolve independently into either related
functional genes, as for example, the embryonic and fetal globin genes, or into
two quite different genes. Finally, a duplicated gene may be nonfunctional, as for
example, the pseudogenes in the globin gene clusters. Presumably, different
selective conditions favor one or the other course.
In this context, the situation with satellites is very striking. Is there in the
sequences an inherent tendency toward amplification greater than that of, say,
the dihydrofolic acid reductase gene or is there positive or negative selective
pressure that operates to maintain multiple copies? Is there a selective force that
dictates a minimal number of copies? Is there a copy number at which additional
amplification is rejected? Is the location at centromeric qnd telomeric positions
influential in determining copy number? It is not sufficient to argue that these

REPEATED SEQUENCES IN MAMMALIAN GENOMES

91

DNA segments e,xist only for their own replication (Orgel and Crick, 1980;
Doolittle and Sapienza, 1980), without asking how they get away with this
profligate reproduction while other sequences do not. What distinguishes a se-
quence that is tolerable in millions of copies from all the other genomic segments
that are not? Is it the inability to be transcribed? Do the satellites provide a
particular environment for genes or special sequences buried within them? The
minimum requirements for centromere function in mitosis and meiosis can be
supplied without millions of tandem repeats, as is evident from the recent work
on yeast centromeres described above. It is conceivable that only one or a few
copies of the satellite sequences are essential to cell function and that, unlike
many other genomic segments, extra copies are tolerable safeguards and collect
by virtue of amplification and the lack of negative selective pressure. This
hypothesis provides a middle ground between the bold suggestion that satellite
has no function at all (Orgel and Crick, 1980; Doolittle and Sapienza, 1980) and
the well grounded assumption that biological systems are efficient.
Another set of questions arises in relation to the heterochromatic location of
satellite. Is late replication in the cell cycle a consequence of heterochromatin
structure or is it dictated by the sequences themselves? Does heterochromatin
structure supply a useful positive function or does it represent a defensive
mechanism whose chief advantage is to protect the genome from the effects of
satellite sequences while still permitting functioning of those sequences at appro-
priate times or places? The latter idea is consistent with other examples of
selective heterochromatin formation, as in X-chromosome inactivation.

IV. Interspersed Repeated Sequences

A. EMERGING CONCEPTS

Interspersion analysis-the measurement of the percentage of denatured DNA
that registers as double-stranded on hydroxyapatite at a fixed low Cot value as a
function of chain length (Davidson et al., 1973)-demonstrated that some highly
repeated sequence families are dispersed among single-copy sequences in
eukaryotic genomes. Two types of interspersion patterns were discerned. The
Drosophila pattern (Manning et al., 1975) is characterized by families of repeat
units several kilobase pairs in length separated by.tens of kilobase pairs of
single-copy sequence. This arrangement has been confirmed by molecular
analysis and several repeated elements in Drosophila are known to be mobile
units (for review see Calos and Miller, 1980; SpradIing and Rubin, 1981). The
Xenopus pattern (Davidson et al., 1973) is characterized by families of repeat
units a few hundred base pairs in length separated by up to a few thousand base
pairs of single-copy sequence. Very few organisms and not even all insects have

92 MAXINE F. SINGER

the Drosophila type pattern but many, including most mammals, are of the
Xenopus type. Several organisms including chickens (Musti et al., 1981) show
interspersion patterns between the two extremes. And within the Drosophila
genome another kind of arrangement, scrambled clusters of different short, mod-
erately repeated sequence elements, occurs (Wensink et al., 1979).
Substantial analysis at the molecular level has now revealed additional com-
plexity. Two different classes of mammalian interspersed and highly repeated
sequences have thus far been distinguished on the basis of size and relative
abundance. Dispersed families with unit lengths under 500 base pairs are found
in as many as hundreds of thousands of copies; they are here termed SINES, for
short interspersed repeated sequences. In some organisms (e.g., humans) the
copy number of the major SINE family is higher than the copy number of some
satellite sequences, thus emphasizing the fact that satellite is not always the most
rapidly reannealing portion of a genome. Other dispersed mammalian families
are several kilobase pairs in length and occur on the order of 104 times (or fewer);
they are here termed LINES, for long interspersed repeated sequences.

B . SHORT INTERSPERSED REPEATED SEQUENCES (SINES)

SINE families often contain more than lol member sequences although smaller
families also exist. The many members in a family are similar enough to hy-
bridize to one another under stringent conditions, but are not identical (Rinehart
et al., 198 1). It is quite possible that different families in a particular species are
related to one another. Although a variety of functions have been suggested
(Britten and Davidson, 1969; Davidson and Britten, 1979; Jelinek et al., 1980)
the physiological significance of these sequences remains unknown. The
strongest available arguments for assuming a functional importance for at least
some members of these families are their marked conservation and the fact that
they are transcribed. The characterized families of rodent and primate SINES
share common features but are, at the same time, quite different.
The repeat units occur in both possible directions within genomic segments;
the significance of the direction, if any, is unclear. This arrangement accounts for
many if not most of the abundant short inverted repeats previously described in
mammalian genomes and is reflected in nuclear RNA molecules that also contain
inverted copies of family members. Transcripts of family members are most
abundant in heterogeneous nuclear RNA but also occur in cytoplasmic RNA.

1. Human Alu Family

The best characterized mammalian SINE family is the Ah family of the human
genome. The presence of a large set of cross-hybridizing short DNA sequences
dispersed throughout most of the human genome and occurring frequently in both
possible orientations was demonstrated initially by renaturation kinetics, hyd-

REPEATED SEQUENCES IN MAMMALIAN GENOMES

93

shear to denature reanneal *.

HumanPfficentalDNA - - -

(100) Lkbp

I

SI nuclease

- - Alu I - gel -
=P 170bp 12Obp 3OObp electrophoresis

-+=+-----

(1.31 - (5)

(3)

I BamH1 linkers

FIG. 8.  The isolation and cloning of the human Alu family (Houck et al., 1979). The cloning
vector was the E. coli plasmid pBR322. The numbers in parentheses indicate the approximate
percentage of total DNA in each fraction.

roxyapatite separation, interspersion analysis, and electron microscopy (Schmid
and Deininger, 1975; Deininger and Schmid, 1976a). Alu family sequences were
first isolated (Fig. 8) (Houck et al., 1979) by renaturing sheared (2 kbp) dena-
tured human placental DNA to a Cot of 68 and removing single-stranded tails
with S1 nuclease. Analysis of the size of the double-stranded products by gel
electrophoresis revealed a broad range of sizes but about 5% of the products were
300 bp in length. More than 60% of the 300-bp-long set of duplexes was cleaved
by AluI into two fragments of about 170 and 120 bp in length, respectively
(Houck et al., 1979): thus the name, Alu family. More precise measurements
now put the reiteration frequency of the family close to 3 x 105 copies or about
3% of the genome (Rinehart et al., 1981). Other families of SINES may also be
interspersed in the human genome (Fritsch et al., 1981; Deininger et al., 1981),
but it seems unlikely that any approach the copy number of the Alu family
(Rinehart et al., 1981).
Direct evidence for the distribution of Alu sequences comes from studies with
long cloned segments of the human genome. Recombinant libraries that comprise
the bulk of the human genome divided into segments 15-20 kbp long, each
inserted into a A-bacteriophage vector, are available (Maniatis et al., 1978; Lawn
et al., 1978). Screening of individual phage in a library by hybridization with a
cloned AZu segment indicates that more than 90% of thk human segments hy-
bridize with Ah (Tashima et al., 1981). Many phage that were selected from a
library for other reasons also contain Alu sequences. There is, for example, an
Ah sequence some 6 kbp downstream from the 3' end of the insulin gene (Bell et
al., 1980); it is the only Alu sequence within about 19 kbp. There are 8 Alu
sequences within the 65 kbp region that contains the entire cluster of P-globin

94 MAXINE F. SINGER

genes (Duncan et al., 1979; Coggins et al., 1980; Baralle et al., 1980; Fritsch et
al., 1980). Eight cloned segments randomly selected from a human library have
a minimum of 9 Alus in a total of 88 kbp (Pan et al., 1981). And 3 Ah segments
are within a 15 kbp segment that contains DNA homologous to the transforming
gene of simian sarcoma virus (Dalla-Favera et al., 1981); all three are within
putative intervening sequences (introns). Altogether this represents 21 Alu se-
quences in a total of 187 kbp of DNA or one AZu sequence every 9 kbp. If a
similar distribution occurs in all the library segments then there are an average of
1-2 Ah family members in each of the 90% that hybridized, approximating the
3 X lo5 copies estimated by other means.
The primary nucleotide sequence of 11 randomly selected and cloned members
of the Alu family is known (Rubin et al., 1980b; Deininger et al., 1981; Pan et
al., 1981). Ten of the 11 are part of a group referred to as BLUR (Rubin et al.,
1980b; Deininger et al., 1981) and were selected from the 300-bp-long frag-
ments obtained from the total genome (Fig. 8). These 10 sequences were used to
construct a consensus sequence (see Fig. 9,l). Although the individual BLUR
sequences differ from each other (and from the consensus) both by insertions,
deletions, transitions, and transversions, they are, on the average, 88% alike.
The alterations are spread around the sequence thus accounting for the strong
cross-hybridizations. In addition, the sequence of four Alu family members from
known positions in the human genome have been determined: one near the
insulin gene (Bell et al., 1980) and one each preceding the -globin gene (Baralle
et al., 1980), the Gy-globin, and the &globin genes (Duncan et al., 1981). All
are approximately 300 bp long and are about 80% homologous to the consensus.
Certain very striking features of Ah family members have emerged from the
sequence analysis. First, the 300 bp unit is essentially a head-to-tail dimer of a
sequence about 135 bp long. One-half of the internally repeated sequence is
interrupted by about 30 extra bp (approximately residues 218 to 250, Fig. 9,l).
Each half is terminated by a stretch of A-T base pairs though this is generally
most striking toward the right end (as written, Fig. 9,l). Within each half is a
14-bp segment (residues 41 to 53 and 176 to 188, in Fig. 9,l) which is similar to
a sequence around the origin of replication of papovaviruses (Jelinek et al.,
1980).
Short direct repeats flank the Ah sequence in each instance studied (Fig. 10);
the BLUR clones do not include flanking sequences because of the way in which
they were isolated. These flanking repeats are not part of Alu itself and are
different in each case examined both in length and structure (Fig. 10). A similar
direct repeat has been found in a sequenced Alu segment from the African green
monkey genome (see below and Fig. 10).

2. Transcription of Human Alu Sequences
The percentage of cytoplasmic RNA that hybridizes to Alu is at least 10-fold
less than that found in nuclear RNA. Heterogeneous nuclear RNA (hnRNA)

REPEATED SEQUENCES IN MAMMALIAN GENOMES

95

1 t I I

1

I GCT GGGCGTG C TGGCTCACA CCTGTAATCC CAGCACTTTG GGAGGCCGAG GTGGGTGGAT
2 GCC GGGCGGG A TGGCTCATG CCTGTAATCC CAGCACTTTG GGAGTTCGAG GCGGGAGGAT
                                            GACTGCCTT
5 CGGGGC TG GAGAGATGGCTCAGC GGTTAAGAGC GCCC
6 GAGGC TG GAGAGATGGCTC GA GGTTAAGAGC ACCA ACTGCTGT

1 t 1 1

I

1 CACCTGAGGT   CAGGAGTTCA      AGACC AGCCT   GGCCAACATG  GTGAAACCCC   GTCTCTACTA
2 CACCTGAGGT   CGGGAGTTCG      AAACC TGC T   GGCCAACATG  GCGAAACCCC   GTCTCTACTA
5 CCAGAGGT   CATGAGTTCA      ATTCCCAGC     AACCA CATG  GTGGCTCACA   ACCATCTGTA
6 TCCAGAGGT   CCTGAGTTCA      ATTCCCAGC     AACCA CATG  GTGGCTCATA   ACAATCTATA

I I I I

5 AAGAGATCTG   ATGCCCTCTT      CTGGTGTATC    TGAAGACAGC  TACAGTGTAC   TTATATATAA
6 ATGAGATCTG   GTGCCCTCTT      CTGGTGTGCA    GATATATATG  GAAGCAGAAT  GTTGTATACAT

t

1 AAAATACAAA   AATTA
2 AAAATACAAA   AATTA
5 TAAATAAATA   AATCTTTAAA AAAAACAAAA CAAAAACAAA AACAAAA
6 AATAAATA   AATAAAATCT TAAAAAAA

, , ,

1 GCCGG      GCGT GGTGGC    GCGCGCCTGT    AATCCCAGCT  ACTCGGGAGG   CTGAGGCAGG
2 GCCG       GTGT GGTGGC    GCATGCCTGT    AGTCCCGGCT  ACTCGGGAG   CTGAGGCAGG
3 CCGG      GCAT GGTGGT    GCATGCCTTT    AATCCCAGC   ACTCGGGAGG   CAGAGGCAGG
4 CCAG      GCATT GGTGGT    ACACACCTTT    AGTCCCAGC   ACTCAGGAGG   CAGAGGCAGG

C

1 1 I I I I

1 AGAATCGCTT GAACCCAGGA GGTGGAGGTT GCAGTGAGCC GAGATCGCGC CACTGCACTC
2 AGAGTTGCTT GAACCT GGA GGTGGAGGTT TCAGTGAGAC AAGATCACAT CACTGCAC
3 CGGATTTCT CAGTTCGAGG C
4 AGGATCACTT GAGTTCAAGA G C

C
T

1

t

1 CAGCCTGGGC AACAGAGCGA
2 CAGCCTGGGC ACAGAGCAA
3 CAGCCTGGTC TTC AGAGT GAGTTCC AGGACACCAG GGCTA CAGAGAAA

                                                     ACAGAGAAA
4 CAGCCTGGTC TACCAGAGTT CCTGAGTT C AAGCCA GGCTAT

T
A

1 1

1

1 GACTCCATCT  C w AAAAAAAAAAAAA
2 CACTCTA    AGAGAGAG AAAAAAAAA ACCCACAAAA AAA
3 CCCTGTCT -  A rich
4 CCCTGTCT    [(A)nN]i

FIG. 9. Comparison of SINES. (1) is the human Ah consensus sequence (Deininger et ai., 1981);
(2) is a cloned monkey Alu sequence (Grimaldi et al., 1981); (3) is the mouse B1 consensus sequence
(Krayev et al., 1980); (4) is the Chinese hamster consensus sequence (Haynes 'et al., 1981); (5) is the
second half of the dimeric SINE found in the intervening sequence of the rat growth hormone gene
(the sequence is reported in Page et a[., 1981 and the homologies were found using the computer
program of Queen and Kom, 1980); (6) is a Chinese hamster sequence (Haynes et al., 1981) which
resembles the rat sequence shown in line 5. The marks above the human Alu sequence (1) indicate
every tenth base pair in that sequence. Only one DNA strand is shown. Each base in each sequence is
shown, in order, in the 5' (upper left) to 3' (lower right) direction. In sequence (4) frequent variations
In the consensus sequence are noted under the line.

96

MAXINE F. SINGER

l.*AAGATTCAClTGllTAG- Ah -AAGATTCAcTTGlTTAG -

2

3. -GCTITG -Ah- GCllTG -

4.-AAAAGAAAC~GGAAAGGA~C~-~G~cTTGG~G~-

-AAAACAA GCAGGAG - Ah --AAAACAA GCAGGAG -

5.-*.GAGACAACAAATCAgag-Bl--GAGACAACAATCAaAt -

FIG. 10.  Direct repeated sequences surrounding members of the primate and rodent SINE
families. Selectedexamples are given. (1) Alu 5' to human Gy-globin gene (Duncan et ai., 1981);
(2) Alu 3' to human insulin gene (Bell et ai., 1980); (3) a monkey Alu (Grimaldi et al., 1981); (4) a
Chinese hamster SINE (Haynes et al., 1981); (5) a mouse B1 sequence (Krayev et al., 1980).
Imperfections in the repeats are indicated by lower case letters. Other examples may be found in the
following references: Elder et al. (1981), Baralle et al. (1980), Haynes et a[. (1981), Page et al.
(1981), and Grimaldi and Singer (1982).

contains high levels of Alu transcripts in molecules ranging from 100 to more
than 5000 nucleotides in length (Federoff et al., 1977; Jelinek et al., 1978,
1980; Elder et al., 1981). At least some of the transcribed Alu sequences are
found in intra- and intermolecular RNA .RNA duplexes, presumably because of
inverted orientations and the transcription of both strands of Alu family mem-
bers. Ah transcripts are found in both polyadenylated and nonpolyadenylated
cytoplasmic RNA (Pan et al., 1981; Elder et al., 1981; Jelinek et al., 1978;
Tashima et al., 1981). The presence of Alu transcripts in polyadenylated RNA
cannot be taken as evidence that the RNAs are polyadenylated by posttranscrip-
tional modification, as are most eukaryote messenger RNAs, because the long
terminal A stretches in the Ah sequence may account for the binding of these
molecules to the oligo(dT)-cellulose used in standard separations (Elder et al.,
198 1). A discrete 7 S cytoplasmic RNA from HeLa cells (uninfected) hybridizes
to Alu DNA and this is the only abundant HeLa cytoplasmic RNA that hybridizes
with Alu (Weiner, 1980). Analysis of hybrids formed between 7 S RNA and
cloned Alu DNA indicates imperfect duplexes suggesting (Weiner, 1980) that 7
S RNA may be encoded by an unusual subset of the Ah family.

Many but not all Alu sequences in cloned human DNA fragments are trans-
cribed in vitro by RNA polymerase III (Duncan et al., 1979; Elder et al., 1981).
The structure of the transcripts is consistent with initiation at the start of the Ah
sequence and termination beyond the end of Alu and the RNA has the sequence
of the DNA strand in Fig. 9,l. Transcriptional control regions are generally
present internal to the coding sequences of genes transcribed by RNA polymerase
111 (reviewed by Duncan et al., 1981). The Alu consensus sequence includes a
segment 5'-GAGTTCPuAGACC-3' (at about position 75) that is similar to a
sequence noted by Fowlkes and Shenk (1980) (5'-GGGTTCGANNCC-3') as im-
portant in control of transcription initiation by RNA polymerase III. This se-
quence does not appear clearly in the second half of the Ah consensus; the

REPEATED SEQUENCES IN MAMMALIAN GENOMES

97

malogous position is interrupted by the extra base pairs. Similarly, termination
of RNA polymerase III transcription of Alu occurs at a run of 4 or more T
residues, as with other polymerase III templates (Duncan et al., 1981). There is
evidence suggesting that 7 S RNA is synthesized in vivo by RNA polymerase III
(reviewed by Weiner, 1980; Zieve, 1981).

3. Alu-like Sequences in Other Primates

The bonnet monkey (Houck and Schmid, 1981) and African green monkey
(Dhruva et al., 1980; Grimaldi et al., 1981) each has an abundant family of
SINES closely similar to human Alu in copy number and sequence. The galago
(Galago crassicaudatus) has a distantly related family of unknown copy number
(Houck and Schmid, 1981). About 75% of recombinant phage representing a
library of the African green monkey genome hybridize with a cloned human Alu
sequence, corresponding to a minimum of 1.8 X 105 copies per haploid genome.
Further, a group of 4 phage, chosen from the library essentially at random with
reference to Alu, contain together at least 8 copies of Alu or an average of one
every 8 kbp of DNA. The sequence of one of the monkey Alu segments was
determined (Fig. 9,2); it is 84% homologous to the human Alu consensus se-
quence and is flanked by short direct repeated segments (Grimaldi et al., 1981).
Among the cloned monkey DNA segments were some in which the Alu sequence
abutted or interrupted African green monkey a-satellite sequences (see Sections
IU,D and IV,B,6).

4. SINES in Rodents

One predominant family of SINES-the AZu family-have been described in
primates while rodent genomes are known to contain several distinct SINE
families of relative similar abundance. This is not to say that the Alu sequences
are the only SINES in primate genomes; others that may be present on the order
of 10 times have already been detected (Deininger et al., 198 1 ; Fritsch et al.,
1981).

Mouse heterogeneous nuclear RNA forms both inter- and intramolecular
double-stranded regions (reviewed by Georgiev et al., 1981). The double-
stranded regions were isolated from intramolecular duplexes by digesting away
single strands with ribonucleases T1 and A and the resulting RNA fragments
were used to detect molecular clones of genomic DNA that hybridize with the
RNA probes. Two distinct families of SINES termed B1 and B2 were isolated.
Members of the two families each occur about 105 times scattered throughout the
genome (Kramerov et al., 1979; Georgiev et al., '1981). These sequences, like
Alu, occur in both orientations along the genome and the inverted configuration
presumably accounts for much of the intramolecular double-stranded
heterogeneous nuclear RNA. The sequences of 3 randomly chosen cloned mem-
bers of the B 1 family are known and were used to construct a consensus sequence

98 MAXINE F. SINGER

(Fig. 9,3) (Krayev et al., 1980); the mismatch between any pair of the three is
less than 8%. Two of the sequenced Bls represent separate units within a single
2.3-kbp-long cloned fragment of mouse genomic DNA.
The B1 consensus sequence is about 130 bp long followed by an A-T-rich
region (A-rich at the 3'-end of the strand shown in Fig. 9,3). Beside the A-rich
region there are other striking similarities between the mouse Bl sequence and
the primate Alu sequences (Pan et al., 1981). A 40-bp sequence that is repeated
in each arm of Alu (residues 21 to 60 and 156 to 195, Fig. 9,l) is highly
conserved in the B1 sequence (Fig. 9,3); this includes the 14-bp segment
homologous to the origin of replication of papovaviruses. B 1 sequences are also
flanked by short direct repeats of DNA (see Fig. IO). Overall B1 can be seen as
analogous to one half of the Alu dimer. However a human Alu sequence (clone
BLUR 8) does not hybridize to mouse DNA under stringent conditions (Shih et
al., 1981).
The B2 family does not cross-hybridize with B1 (Kramerov et al., 1979).
Other evidence indicating multiple families of abundant mouse SINES is seen in
the fact that three separate, noncross-hybridizing SINES were discovered in-
terspersed among the 65-kbp-long mouse P-globin gene cluster; each of the three
occurs twice or more within the segment (Haigwood et al., 1981). The distribu-
tion of SINES in the P-globin gene cluster of two different Mus musculus strains
(BALB/c and C57BLhO) appears to be essentially the same (Haigwood et al.,
1981). The three SINE families were labeled a, b, and c (Haigwood et al., 1981)
and it is not known if any of them are related to the previously described B 1 and
B2. Cloned members of the a and b families hybridized with essentially all the
phage in a mouse genomic DNA library. Thus, there are at least 2 X 105 separate
members in each of these families assuming that the inability to cross-hybridize
extends to all members of each family. It is possible that mouse SINE family
members are interspersed in mouse satellite (Haigwood et al., 1981).
Five different SINE families occur at 20 positions within the 44-kbp segment
containing the genes for the P-globins of rabbits (Shen and Maniatis, 1980;
Fritsch et al., 1981). Only one family, called C, hybridizes (weakly) to a human
Alu sequence under nonstringent conditions. C sequences, like Ah, are trans-
cribed by RNA polymerase I11 in vitro. Members of SINE families were found
buried in long cloned segments of Chinese hamster DNA. The clones were
selected because they hybridized with double-stranded heterogeneous nuclear
RNA (Jelinek, 1978; Haynes et al., 1981). Thirty-seven percent of the phage in a
Chinese hamster genomic DNA library hybridized. A large proportion of the
cloned segments had more than one hybridizing region and many of these oc-
curred in opposite orientations. The sequences of 6 such regions were determined
and a consensus derived from 5 of them (Haynes et ul., 1981) (Fig. 9,4). The
similarity with the mouse B1 consensus sequence is evident. One of the 6 (Fig.
9, 6) differs markedly from the others after the first 60 base pairs, but is, there-

REPEATED SEQUENCES IN MAMMALIAN GENOMES

99

fore, homologous to a rat sequence (see below). Like Alu and B1, the Chinese
hamster SINES are surrounded by short direct repeats of DNA (Fig. 10).
Much less is known about the situation in rats. Houck and Schmid (1981)
reported the presence of abundant 300-bp-long segments in reannealed rat DNA
that was treated with S1 as outlined in Fig. 8 for the original isolation of human
Alu sequences. One repeated DNA segment occurs within an intron of the rat
growth hormone gene and again after the end of the last exon in the gene (Page et
al., 1981). The sequence of the segment in the intron (Fig. 9, 5) includes two
direct repeats of a segment about 200 base pairs long. This unit may represent a
rat SINE family. It is, as noted above, similar to one Chinese hamster sequence
(see Fig. 9, 5 and 6). A repetitive sequence has also been identified downstream
from the rat insulin I gene (Bell et al., 1980); it does not hybridize with a human
Alu probe.

5. Transcription of Rodent SINES

As already described, the mouse B1 and B2 sequences are abundantly trans-
cribed; copies of both strands of the sequence occur in heterogeneous nuclear
RNA (Kramerov et al., 1979). A unique version occurs in mouse cytoplasm as a
discrete 4.5 S RNA (Harada and Kato, 1980); this sequence differs from the
consensus sequence derived from three cloned members of the B1 family
(Krayev et al., 1980). The 4.5 S RNA hybridizes to both cytoplasmic and
nuclear polyadenylated RNA (Jelinek and Leinwand, 1978). It is striking that the
4.5 S mouse sequence is more similar (3 differences out of about 90 nucleotides)
to a 4.5 S cytoplasmic RNA of the Chinese hamster (see below) than are the
mouse and Chinese hamster consensus DNA sequences to one another (see Fig.
9). One, or a few members of these families may be the functional genes for 4.5
S RNA in each of the species, and if so, that gene has been markedly conserved
(Haynes et al., 1981). Both the mouse and Chinese hamster 4.5 S RNAs are
copies of the same DNA strand (sequence equivalent to the DNA strands in Fig. 9).

6. Are SINES Functional?

As a background, it is interesting to recall proposals suggesting that highly
repeated dispersed sequences may be without function (Orgel and Crick, 1980;
Doolittle and Sapienza, 1980) and also disagreement concerning those proposals
(Cavalier-Smith, 1980; Dover, 1980; T. F. Smith, 1980; Orgel et al., 1980;
Dover and Doolittle, 1980). Specific functions that have been suggested include
the control of gene expression (Britten and Davidson, 1969; Davidson and Brit-
ten, 1979; Jelinek et al., 1980), perhaps by involvement of transcripts of SINES
in the maturation of messenger RNA (Georgiev et al., 1981; Jelinek et al., 1980;
Zieve, 1981; Lerner and Steitz, 198l), and service as origins of DNA replication
(Jelinek et al., 1980; Georgiev et al., 1981). The cytoplasmic 4.5 S RNA of
rodent cells and the 7 S RNA of human cells appear to represent abundant

100 MAXINE F. SINGER

transcripts of a particular member(s) of the respective SINE families. At least
these SINE family members are likely to be functional genes. It is striking that
the 4.5 S RNA of the hamster and mouse are identical in all but 3 of 90
nucleotides (Harada and Kato, 1980; Haynes et al., 1981). They are both found
in small nuclear ribonucleoprotein complexes, as well as cytoplasm and recent
speculations regarding their function are reviewed by Zieve (1981) and Lerner
and Steitz (1981). Altbough the 4.5 S RNAs have 5'-terminal triphosphates,
consistent with in vivo transcription by RNA polymerase 111, it is not clear to
what extent RNA polymerase I1 transcripts that include SINES also contribute to
homologous sequences in heterogeneous nuclear or cytoplasmic RNA.
Some clues regarding SINE function might be expected from a comparison of
their distribution around corresponding genes in different organisms. Extensive
data are available for the &globin gene clusters of mouse (Haigwood et al.,
1981), rabbit (Shen and Maniatis, 1980; Hoeijmakers et al., 1980; Fritsch et al.,
198l), and human (Fritsch et al., 1981) and the a-globin cluster of humans
(Fritsch et al., 1981). Within each cluster are several coding regions, one or
more pseudogenes, and various SINES. In the human p-cluster 5 genes are
expressed in a timed sequence: E in embryos, Ay and Gy during fetal develop-
ment, and 6 and /3 during adulthood. In the rabbit, two genes are expressed in
embryos and one in adults, while in the mouse, two are expressed in adults and
one in embryos (Jahn et al., 1980). Both rat and human insulin genes (Bell et
al., 1980) have a SINE following the end of the last exon, although at different
distances. There is no equivalent to the SINE within one intervening sequence of
the rat growth hormone gene in a corresponding human gene (Page et al., 1981).
While some patterns can be discerned (e.g., in the rabbit the C-SINE family is
clustered in the region of embryonic genes and in the human @globin cluster no
Alu occurs between simultaneously expressed genes) and may prove to be mean-
ingful, no definitive statements can be made at this time.

Another interesting possibility is that some SINES are mobile or transposable
sequence elements analogous to those described in yeast and Drosophila (re-
viewed by Calos and Miller, 1980; Spradling and Rubin, 1981). Mobile elements
are flanked by short direct repeats of sequences that are not part of the element
itself. In the case of characterized mobile elements the repeat lengths are charac-
teristic of the element and constant, and the duplications reiterate the target site at
which insertion occurred. Similarly, integrated DNA copies of RNA tumor virus
genomes in birds and mammals are flanked by such short direct repeats (re-
viewed by Temin, 1980). These viral inserts as well as several well characterized
mobile elements in bacteria, yeast, and Drosophila share other structural fea-
tures. These include direct terminal repeats of several hundred base pairs at the
two extremities of the movable element itself as well as a total length of several
kilobase pairs. Also eukaryote mobile elements do not typically have the long
terminal stretch of AT base pairs that is a common feature of mdmmalian SINES.

REPEATED SEQUENCES IN MAMMALIAN GENOMES

101

However, a zecently discovered movable Drosophila element does have notable
similarities to the mammalian SINES. This element, referred to as lOlF (Dawid
et al., 198l), has no long terminal repeats, is flanked by short direct repeats of
the target site, and contains a 3'-terminal poly(A) segment 18 nucleotides long.
Unlike the mammalian SINES, lOlF is about 4 kbp in length.
Several observations are consistent with the possibility that SINES are mobile
in mammalian genomes, at least in evolutionary time. First, there is no counter-
part in the human gene to the SINE within the intervening sequence of the rat
growth hormone gene, although the genes themselves are likely to be homolo-
gous and the introns similarly placed. Thus, assuming a common ancestral se-
quence, the SINE was either lost during evolution of the human gene or acquired
during evolution of the rat gene. Second, the very different distributions of
SINES in the clusters of globin genes (comparing rodents with primates or the a-
and &clusters of humans) are consistent with mobility. Third, Alu sequences are
found within African green monkey a-satellite segments (Grimaldi et al., 1981).
Because the Alu sequence is highly conserved and satellite was amplified after
separation of the monkey and human evolutionary lines, the Alu sequence was
probably acquired after amplification of the satellite. Finally, the direct repeats
surrounding mammalian SINE segments (Fig. 10) are reminiscent of the target
site duplications that flank mobile elements. Very recently the primary nuc-
leotide sequence of an African green monkey a-satellite segment that is inter-
rupted by an Alu family member was determined. A 13-bp-long repeat surrounds
the Alu and this is a duplication of the a-satellite sequence at the point of
interruption (Grimaldi and Singer, 1982).
Calos and Miller (1980) give an excellent summary of the possible significance
of mobile elements in eukaryotes, emphasizing their potential for the generation
of genetic diversity. The following additional point may be important, in view of
the suggestions that highly repeated sequences have no function at all. A mobile
element may generate diversity with a potential for selective advantage, but it can
also generate disadvantage if it moves into an essential gene. Mutation by mov-
able elements has been demonstrated in yeast (Roeder and Fink, 1980) and
Drosophila (reviewed in Spradling and Rubin, 1981). The high frequency of
mutation caused by the presence of large numbers of movable elements within a
mammalian genome might have proven intolerable and been selected against,
unless it was counterbalanced by some positive functional advantage.
Finally, the suggestion (Jelinek et al., 1980; beorgiev et al., 1981) that
SINES may serve as origins for DNA replication should be considered. The basis
for the suggestion is the presence in SINES of a short (14 bp) homology to a
sequence associated with the origin of replication of murine and primate
papovaviruses. Georgiev et al. ( 198 1) describe some preliminary experiments
that are consistent with this suggestion. However, in papovavirus genomes this
region is part of a complex control region and may be involved in the control of

102 MAXINE F. SINGER

transcription as well as replication. Only additional experiments will resolve
these questions.

C. LONG INTERSPERSED REPEATED SEQUENCES (LINES)

Dispersed repeated sequences several kilobase pairs in length (LINES) have
been described in both rodents and primates. Several LINE families may eventu-
ally be found in eich genome. As yet none is completely characterized.

1. Primates

Several groups of investigators appear to have discovered the same family of
LINES, called here the Kpn-LINE family (Adams et al., 1980; Kaufman et al.,
1980; Schmeckpeper et al., 1981; Maio et al., 1981b; Manuelidis, 1981b;
Rogers, 1981; J. C. Rogers and C. Milliman, unpublished; G. Grimaldi and M.
F. Singer, unpublished). A segment about 6.4 kbp long and estimated to be
repeated 3 to 5 thousand times in the human genome was found 3 kbp
downstream from the 3' end of the human @-globin gene (Adams et al., 1980;
Kaufman et al., 1980). This and several other somewhat divergent randomly
selected members of the family were cloned from a human library. Portions of
this LINE hybridize under smngent conditions with a cloned 2.8 kbp KpnI
fragment of African green monkey DNA; this fragment was found interrupting
and abutting a-satellite and was independently identified as a portion of a
monkey LINE found in thousands of copies in both human and monkey DNA (G.
Grimaldi and M. F. Singer, unpublished). Other members of the monkey family
diverge in such a manner that the sequences in the 2.8-kbp KpnI fragment are
divided into two KpnI fragments, 1.2 and 1.5 kbp in length, respectively. The
2.8-kbp fragment hybridizes to both monkey and human genomic KpnI frag-
ments 1.2, 1.5 (1.6), 2.8, 3.4, and 4.6 kbp long; it also hybridizes to monkey
and human HindIII segments 1.9 and 2.6 kbp long. One member of a set of
abundant 1.9-kbp human HindIII fragments was cloned separately and shown to
hybridize to KpnI fragments of the same size (Rogers, 1980; J. C. Rogers and C.
Milliman, unpublished). Independent work by Schmeckpeper et al. (1981) iden-
tified a LINE family that also hybridized with the same size classes of HindIII
and KpnI fragments from the human genome. The LINE family was located on
both X and autosomal chromosomes. The Kpn-LINE family appears to be common
among primates since digestion of total DNA from several species yields the
distinctive set of KpnI bands (Maio et al., 1981b).

The dispersed organization of the KpnI-LINE family is inferred from (1) its
presence on human X and autosomal chromosomes (Schmeckpeper et al., 1981;
Manuelidis, 1981b), (2) its distribution in the phage of the monkey (G. Grimaldi
and M. F. Singer, unpublished) and human (Adams et ai., 1980) libraries, and

REPEATED SEQUENCES IN MAMMALIAN GENOMES

103

(3) the absence of any "ladders" upon restriction endonuclease cleavage (all
papers already mentioned).
Five percent of the bacteriophage comprising an African green monkey
genomic library (McCutchan et al., 1981) hybridized with the cloned 2.8 KpnI
fragment, consistent with about 12,000 dispersed copies of the sequence. Some
estimates for the human genome (Maio et al., 1981b; Rogers, 1981) are similar,
although these numbers are markedly higher than the 3-5 thousand copies esti-
mated by Adams et al. (1980). J. C. Rogers and C. Milliman (unpublished) have
obtained data suggesting that the copy number of the Kpn-LINE family may
vary from individual to individual.
It is already clear that there is extensive divergence among the members of the
Kpn-LINE family although the various members cross-hybridize. Both the
cloned human segments (Adams et al., 1981) and cloned monkey segments (G.
Grimaldi and M. F. Singer, unpublished) show restriction endonuclease site
variability. Also, it is evident from digests of total genomic DNA hybridized to
cloned probes that the abundance of different variants is very different in
monkeys and in humans (G. Grimaldi and M. F. Singer, unpublished). Data
suggesting that Kpn-LINE family members cross-hybridize with a-satellite
(Maio et al., 1981b) are puzzling since other investigations give no indication of
such a relation (McCutchan et al., 1981; G. Grimaldi and M. F. Singer, unpub-
lished; Manuelidis, 1981b). It is probable that the uncloned a-satellite probes
were contaminated with Kpn-LINES.

2. Rodents

Several groups of workers have obtained data on what appears to be a single
abundant LINE family in mice, called here the EcoRI-LINE family. Between 1
and 3% of the Mus musculus genome is converted to a set of fragments 1.3 kbp
in length upon digestion with EcoRI; this is equivalent to as many as 50,000
copies per haploid genome (Horz et al., 1974; Cheng and Schildkraut, 1980;
Heller and Amheim, 1980; Manuelidis, 1980; Brown and Dover, 1981;
Meunier-Rotival et al., 1981). The 1.3-kbp segment is frequently if not always
contained within repeat units that are at least 5.6 kbp in length (Meunier-Rotival
et al., 1981; Brown and Dover, 1981). Although the sequence itself is conserved
among the members of the family, subgroups of family members show marked
divergence in restriction endonuclease sites (Brown and Dover, 198 1; Meunier-
Rotival et al., 1981). EcoRI-LINES probably occ& on many mouse chromo-
somes. Homologous families that largely retain the internal EcoRI segment have
been detected in other species of Mus, in Apodemus genomes, and in rats and
Chinese hamsters (Heller and Amheim, 1980; Brown and Dover, 1981). The
relative abundance of different variants of the EcoRI-LINE family, however,
differs in most of these species.

104 MAXINE F. SINGER

Preliminary data on other possible families of LINES have been published.
One, estimated to be repeated about 4 x 104 times, comprises part of the
nontranscribed spacer region in the array of mouse genes for ribosomal RNA and
also occurs flanking genes for the constant region of mouse heavy chain im-
munoglobulin (Amheim et al., 1980). This family is not homologous to the
1.3-kbp EcoRI segment of the EcoRI-LINE family. Two relatively abundant
nonsatellite fragment sets 1.5 and 1.7 kbp long, respectively, and estimated to
comprise together about 0.2% of the genome are produced by EcoRII cleavage
(Manuelidis, 1980); neither Qne hybridizes with the other. Preliminary evidence
indicates the presence of an abundant LINE family in the Kangaroo rat (Liu and
Lark, 1981).

3. Are LINES functional?

The discovery of LINE families in mammals is recent and there is very-little
information available regarding function. Adams et al. (1980) found no tran-
scripts homologous to the human Kpn-LINE farriily in bone marrow cells and
Manuelidis (1981b) also reports negative preliminary experiments. There is no
information available regarding the possibility that LINES are mobile in mamma-
lian genomes.

E. AMPLIFICATION AND DISPERSION

SINE family members are dispersed between genes, in introns, and within
satellite DNA sequences. The SINES are also highly conserved within genomes
and between relatively closely related mammalian groups such as members of the
genus Mus or old world monkeys and man. This conservation is in marked
contrast to the very different, noncross-hybridizing (under stringent conditions)
satellites of, for example, various old world monkeys and man. On the other
hand, when the SINE families of primates and rodents are compared, major
differences are apparent even though the repeat units have some striking
homologies. There are markedly different multiplicities of different SINE
families in different genomes. Further, the B1-SINES of the mouse are, for
example, about 130bp in length while the Alu-SINES of primates are more than
twice that length. At some time after the rodent and primate evolutionary lines
separated, one or the other or both lines acquired distinct families of SINES. The
mechanism by which the acquisition occurred necessarily involved amplification
into many dispersed positions. Providing experimental data relevant to an under-
standing of that mechanism(s) is a major challenge for future research. The
conceptual problems have already been discussed (Scherer and Davis, 1980; Pan
et al., 1981; Dover, 1981; Baltimore, 1981; Grimaldi et al., 1981). Unequal
crossing-over, which can account for the amplification of satellite DNA (Section
IIIJ), is less satisfactory with regard to amplification and dispersal of SINES.

REPEATED SEQUENCES IN MAMMALIAN GENOMES

105

The process balled gene conversion may provide a better model. Gene conver-
sion is a nonreciprocal recombination; a DNA sequence is duplicated at a (partly)
homologous distinct genomic site without being lost from the original "donor"
site. Both intrachromosomal (Scherer and Davis, 1980) and interchromosomal
(Jackson and Fink, 1981; Klein and Petes, 1981) gene conversion have been
demonstrated in yeast. Multiple gene conversions might account for the mass
change from one SINE family to another partly homologous SINE family as well
as the maintenance of homogeneity in an established SINE family. The different
relative reiteration frequencies of SINE families might reflect differential rates of
gene conversion in different species, but might also reflect more fundamental
distinctions between genomes. However, by itself gene conversion does not
easily explain the initial or any continuing dispersion of SINE family members
into the genome. Dispersion could be explained if SINES prove to be mobile (or
transposable) elements, but the mechanism of transposition would have to be
such that the donor sequence remains in its original site in addition to being
duplicated at a new one (as in gene conversion). The transposable elements of
prokaryotes appear to act in just such a way (reviewed by Calos and Miller,
1980).
Similar considerations apply to the amplification and dispersion of LINES.
Also, it will be important to understand the processes that maintain LINES in
smaller numbers of copies than SINES. Recent data suggest that LINES may be
associated with major components of mouse DNA (nonsatellite) that are separa-
ble by density gradient centrifugation (Soriano et al., 1981; Meunier-Rotival et
al., 1981). Perhaps, unlike SINES, LINES are restricted to particular genomic
regions. Second, the extensive polymorphism of LINES within a given species is
interesting. Does it reflect a fluid, continually changing population or is it a fixed
distribution? And what, if any, are the functional consequences of the
polymorphism?

V. Concluding Remarks

The precise insights into the molecular structure and organization of highly
repeated sequences that are afforded by modem techniques are rewarding but
frustrating. This is especially so in mammals compared, for example, to
Drosophila (Spradling and Rubin, 1981), because of the difficulties in wedding
genetics or cytogenetics with molecular analysis. New experimental approaches
to the question of function are essential. One such approach, the search for
proteins that specifically bind repeated sequences, has been urged by Brutlag. A
protein that interacts specifically with one satellite from Drosophila melanogas-
ter has been described (Hsieh and Brutlag, 1979b), but unfortunately this ap-
proach has not yet been applied to other organisms. Recent progress in elucidat-

106 MAXINE F. SINGER

ing the structure of mammalian kinetochores provides interesting possibilities for
studying proteins that might bind to satellites (Ris and Witt, 1981; Brenner et al.,
1981). Another avenue yet to be fully exploited involves analysis of the effects of
repeated sequences on replication and transcription of small, constructed
genomes. Genomes for use in mammalian cells have been designed (Hamer,
1980; Mulligan and Berg, 1980) and recombinant DNA techniques permit the
introduction of repeated sequences. It is to be hoped that an emphasis on function
will develop in the near future.

ACKNOWLEDGMENTS

I am grateful to the following investigators who provided me with preprints: Molly Fitzgerald-
Hayes, John Carbon, Joe Maio, Giorgio Bernardi, Prescott Deininger, K. G. Lark, Carl Schmid,
Laura Manuelidis, Hans Zachau, A. Plucienniczak, John Rogers, Rolf Streeck. My colleagues
Antonella Maresca, Giovanna Grimaldi, Theresa N. H. Lee, and Ronald E. Thayer, kindly and
critically reviewed the manuscript.

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NOTE ADDED IN PROOF

The mouse interspersed repeated family described by Arnheim et al. (1980) is probably a SINE
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