ANNUAL REPORT OF THE

   LABORATORY OF BIOCHEMICAL GENETICS

NATIONAL HEART, LUNG, AND BLOOD INSTITUTE

October 1,  1989 through September 30, 1990

Previously, we cloned three neighboring Drosophila homeobox genes

designated NK-1, NK-3, and NK-4 that reside in the right arm of

the third chromosome at bands 93El-5, and a fourth homeobox gene,

NK-2 that resides in the X chromosome at bands lCl-5.

The nucleotide sequences of approximately 3.5 kb of NK-2 cDNA and

genomic DNA were determined.  The deduced amino acid sequence of

NK-2 protein consists of 723 amino acid residues.  NK-2 is a basic

protein, with regions rich in alternating acidic and basic amino

acid residues, regions rich in seryl-, threonyl-, and

prolyl-residues, repetitive alanine residues, PEST sequences,
which specify rapid turnover of protein, an acidic domain, a

homeobox domain, and an alternating repeat of histidine and

alanine residues.  Northern analysis revealed one species of NK-2
poly A+ RNA, 3.2 kb in length,  which is most abundant in 3-6 hr

embryos.  NK-2 mRNA was detected at all later stages of embryonic
development and in adult flies,  but less NK-2 mRNA was detected in

larvae and pupae.  In situ hybridization analysis showed that
expression of the NK-2 gene is initiated at the cellular
blastoderm stage in the ventrolateral portion of the embryo, which
later in development, gives rise to the nervous system.   In 12 hr
and older embryos expression of the NK-2 gene was found both the
Central nervous system and in cells associated with the midgut.


The nucleus of a fertilized DrosoDhila egg divides thirteen times

during the first 130 minutes of development to form the

blastoderm, which is a single cell with five thousand nuclei.
During the next forty minutes of development plasma membranes are
laid down around each nucless resulting in the  formation of a

five thousand cell embryo termed the cellular blastoderm.   As the

cellular blastoderm is forming,  the nuclei in the ventrolateral

portion of the blastoderm become committed to the neuroectodermal

pathway of differentiation.  This formation of the neuroectodermal
anlage is the first step in the developmental program that leads

to the formation of the nervous system.  Both the initial time of
expression of the NK-2 gene at the cellular blastoderm stage of
development and the initial pattern of expression of NK-2 coincide
exactly with the appearance of the neuroectodermal anlage.   Since
homeobox proteins are known to be regulators of gene expression,
we predict that NK-2 protein activates the gene program that
commits cells to the neuroectodermal pathway of development and
thereby initiates the developmental program for the formation of

the nervous system.

Both genomic and cDNA NK-1 clones were sequenced and approximately
7500 nucleotide residues of the NK-1 gene were determined.   The

NK-1 gene contains 4 exons; the last intron is located within the
region of the DNA that codes for the third helix of the homeobox
protein at the same site as the corresponding introns of labiaL,
Abdominal-B, and Distal-less horn&ox genes.  NK-1 protein is
comprised of 661 amino acid residues and contains repetitive,


alternating histidyl-prolyl residues termed a gajred repeat,

repetitive histidyl-glutamyl residues,  repetitive alanyl residues,

a highly acidic domain before the homeobox, and PEST sequences.
One major poly A+ RNA transcript, 2.9 kb in length, was detected

in 6-12 hr embryos and thereafter during embryonic development,

but not in O-3 hr embryos.  Ln situ hybridization analysis of

embryo sections and whole embryos showed that the expression of

the NK-1 gene starts in 7.5-8.0 hr embryos in the ventral nervous

system and ultimately results in a bilaterally symmetric,

segmentally repeated pattern that consists of a of small subset of

neurons on each side in each thoracic and abdominal segment and

some unidentified cells in the head.  The NK-1 gene also is

expressed in a similar pattern in a small subset of striated

muscle cells in each thoracic and abdomenal segment.

The nucleotide sequence of Drosophila, NK-1 homeobox DNA was used

to clone genomic DNA from monkey, rat,  .Xenoous, and salmon DNA.

Nucleotide sequence analysis showed that the deduced amino acid

sequences of the homeobox domains of monkey and rat are identical

to that of the Drosophila NK-1 homeobox,  and that salmon and

XenOWuS homeoboxs are novel home&ox f that differ from NK-1 by 4
and 9 amino acid residues, respectively.

NK-3 and NK-4 are neighboring homeobox genes that are separated by

only 7.5 kb.  Approximately 3.6 kb of cloned NK-3 cloned cDNA and

genomic DNA were sequenced.  The NK-3 gene contains a short intron


4

and codes for a homeobox protein 374 amino acid residues in

length, which contains charged regions rich in acidic and basic

amino acids, and a region rich in serine, threonine, and proline

residues.  Northern analysis of NK-3 revealed one species of poly
A+ RNA, 1.6 kb in length, that is expressed transiently in 6 to 12

hr embryos but not thereafter.  By FB situ hybridization NK-3 mRNA

was shown to be  expressed in a bilaterally symmetric, segmentally
repeated pattern in the visceral mesoderm of Drosowhila embryos.

Sequence analysis of NK-4 cDNA and genomic DNA clones show that
the NK-4 gene has 2 introns and that the deduced NK-4 protein is
371 amino acid residues in length.  NK-4 is a basic protein with

CAX repeats, a homeobox domain, glutamine repeats (M repeats), and
regions rich in serine, threonine, and proline.  One major species
of NK-4 poly A+ RNA was detected, 1.7 kb in length, which first
appears in 3 - 6 hr Drosowhila embryos,  is less abundant in 6 - 12

hr embryos,  and was not detected thereafter.  NK-4 was found by h
situ hybridization to be expressed only in cephalic, thoracic, and
abdominal mesodermal tissue starting at gastrulation (3 hr) when

the mesoderm first appears.  NK-4 mRNA was not detected after 7.5

hr of embryonic development.

Short segments of mouse genomic DNA that correspond to homeobox
regions of homeobox genes were selectively amplified by the
polymerase chain reaction and were cloned.  Nucleotide sequence
analysis showed that some of the DNA clones correspond to novel

mouse homeobox genes.  A mouse genomic DNA library with inserts in


the 15 kb range then was screened using the small  DNA inserts

that had been identified as novel mouse homeobox genes as probes.

Genomic DNA clones, were obtained that correspond to four novel

mouse homeobox genes.  The nucleotide sequences before and after

the homeobox regions of two of the genomic DNA clones (CL-8B and

CL-101) were determined.  RNA transcripts of portions of CL-8B DNA

then were used as probes to screen a mouse embryo cDNA library and

a CL-8B cDNA clone was obtained.  Further work is needed to

determine the structures of these homeobox genes and the time and

sites of expression of the genes in mouse embryos and adults.

A subfamily of homeobox genes has been reported that code for

proteins that contain a highly conserved domain, termed a Pou-box,

before the homeobox.  During the past year, two novel species of
Pou-box-homeobox cDNA were cloned and the Pou-box-homeobox regions

were sequenced.  Both of the novel cDNA's correspond to species of
poly A+ RNA that are expressed in adult mouse brain.

A method of selection for cloned mouse genomic DNA fragments that
contain functional enhancer or promoter sequences was developed
that is based on the observation that the synthesis of polyoma
virus DNA in mouse cells is dependent upon viral enhancer or

promoter sequences that also are needed for activation of

transcription of polyoma genes.  An &. c_ol-mammalian cell shuttle

vector, pPyE0, was used that contains the P-lactamase gene and
origin of replication from pBR322, and polyoma viral DNA with a
library of mouse genomic DNA fragments inserted in the vector in


place of the polyoma enhancer region.  Thus,  recombinant plasmids

with DNA inserts that contain enhancer or promoter sequences were

expected to replicate in mouse  cells,  but not recombinants that

lack such sequences. Neuroblastoma, glioma, myoblast, or
fibroblast cell lines were transfected with plasmid DNA and
incubated for several days to allow plasmid replication.   Then

plasmid DNA was recovered, purified,  and cloned in E. u.

Thousands of recombinants were recovered from mammalian cells and

all of the cloned DNA inserts that were tested had enhancer

activity when ligated to a reporter gene.  The results show that

the enhancer selection method yields many kinds of cloned enhancer

sequences.  Marked differences also were found in the activities
of some cloned sequences in stimulating the expression of a

reporter gene in different cell types.

Previously, we showed that prolonged elevation of cyclic AMP
levels of NG108-15 neuroblastoma-glioma hybrid cells and other
neuroblastoma cell lines results in the appearance in cells of
electrically excitable ion channels, and that storage vesicles for
acetylcholine increase in abundance, and the number of synapses of
NG108-15 cells with striated muscle cells increases.   The increase
in synapses is due to a CAMP-dependent acquisition by cells of
functional L-type voltage-sensitive calcium channels that are
required for stimulus-secretion coupling in NG108-15 cells.   cDNA
clones were obtained that correspond to mRNA from two
voltage-sensitive calcium channel al-subunit genes from rat brain.
Northern analysis revealed that poly A+ RNA corresponding to an


7

al-subunit of a voltage-sensitive calcium channel markedly

increased in abundance when NG108-15 cells were incubated for

several days under conditions that increase cellular levels of

CAMP.

In addition, cDNA clones were obtained that correspond to species
of NG108-15 poly A+ RNA that increase in abundance when cells are

treated with compounds that elevate cellular CAMP.   Six kinds of

w cDNA clones were identified by nucleotide sequence analysis.
Four of the cDNA clones correspond to different portions of mouse

mitochondrial DNA; i.e., pNG-10 corresponds to RNA transcribed

from the mitochondrial DNA displacement loop which contains
promoters for the transcription of mitochondrial heavy- and light-

chain DNA, the origin of replication for mitochondrial heavy chain

DNA, and other regulatory signals.  pNG-32 cDNA corresponds to
mRNA transcribed from the mitochondrial ATPase 6 gene, which codes
for a protein that is part of the proton pump-ATP synthase

complex.  pNG-37 corresponds to mRNA transcribed from the
mitochondrial cytochrome oxidase I; whereas, pNG-47 corresponds to
mitochondrial 12s ribosomal RNA.  Treatment of NG108-15 cells with

dibutyryl CAMP for 5 - 8 days results in two to five fold
increases in RNA transcripts from both the mitochondrial heavy and
light strands of DNA and in a two to three-fold increase in the

amount of mitochondrial DNA.  Similar results were obtained when
adenylate cyclase of NG108-15 cells was activated with

prostaglandin El, mediated by PGEl receptors, or by forskolin. In

addition, pNG-64 cDNA was shown to correspond to secretogranin I,


a secretory vesicle protein.  Poly A+ RNA for secretogranin I was

found to increase three-fold in abundance due to treatment of

cells for 5 days with dibutyryl CAMP.  cDNA clone pNG-57 was shown

to correspond to mRNA for ribosomal protein S-10.

In previous studies CAMP and phorbol esters that activate protein

kinase C were shown to synergistically elevate neuropeptide Y mRNA

levels 20 to 200-fold in 4 to 24 hr in PC12 rat pheochromocytoma

cells.  Treatment of cells with nerve growth factor (NGF) results

in 40 to loo-fold increases in neuropeptide Y mRNA within 1 to 6

days.  Glucocorticoids biphasically modulate the stimulations by

NGF, potentiating early (3-10 hr) effects of NGF, but inhibiting

later stimulations (l-6 days).   The results of nuclear run-on

transcription assays showed that the apparent rate of neuropeptide

Y gene transcription in PC12 cells is altered by NGF, NGF plus

dexamethasone,  and CAMP plus phorbol ester.   The effects of these

compounds on neuropeptide Y mRNA stability were less significant.

These results show that neuropeptide Y gene transcription is

controlled by multiple, potentially interacting regulators.

To identify regions upstream from the neuropeptide Y promoter that

regulate gene expression, portions of the upstream region of the

neuropeptide Y gene were ligated to plasmids that contained the

chloramphenicol acetyltransferase (CAT) gene.   In transient

expression assays,  constructs that contained 144 and 671

nucleotide residues of upstream sequence were weakly responsive to

CAMP elevation by forskolin or to NGF, but no enhancement by


9

phorbol ester was noted.  The responses were much less than were

observed with the endogenous gene,  suggesting the additional

sequences or variables are important.

Glucocorticoids such as dexamethasone potentiate proenkephalin

gene expression in several systems including rat brain. We

previously showed that glucocorticoids and CAMP synergistically

elevate proenkephalin mRNA levels.  Nuclear run-on transcription

assay results show that the proenkephalin gene transcription rate

was not altered by dexamethasone alone, was weakly stimulated by

CAMP, and was persistently stimulated by dexamethasone and CAMP

together (3-6-fold over l-24 hr). To search for cooperative
glucocorticoid and CAMP regulatory elements, chimeric plasmids
were constructed containing the CAT reporter gene under the
control of upstream proenkephalin gene sequences in C6 glioma

cells transfected with these constructs.  Full stimulation of CAT

activity by CAMP required sequences between residues -145 and
-192, which are beyond the previously described human

proenkephalin CAMP responsive site.  Unexpectedly, dexamethasone
did not stimulate expression but reduced CAMP stimulated CAT

activity; the full negative effect required sequences between -435

and -1000.  A DNA fragment from the 5" upstream region of a rat
proenkephalin gene was inserted into a promoterless CAT vector and
a Rous sarcoma virus-transformed quail embryo retina cell line was

transfected with the DNA.  Treatment of the transfected cells for
24 hr with forskolin, prostaglandin El and theophylline, or
retinoic acid resulted in 12-, 6-,  and $-fold increases in CAT


10

expression,  respectively.  In addition,  the transactivation

protein tax1 of human T-cell leukemia virus I was found to
activate the proenkephalin gene promoter and resulted in a
3-g-fold increase in CAT expression in C6 glioma cells
transfected with the chimeric rat proenkephalin regulatory

region-CAT DNA.

Ankyrin, a peripheral membrane protein that links certain integral
membrane proteins to the spectrin-actin membrane cytoskeleton, was
shown by immunofluorescence and immunogold labeling and electron
microscopy to be concentrated at the triad junctions of striated
muscle cells formed by the transverse tubules and the sarcoplasmic

reticulum.  Ankyrin thus may play a role in organizing the

structure of the triad, or may be involved in linking the triad
membranes to the muscle cytoskeleton at specific sites along the

sarcomere.

Mice with the mdg/mdg genotype die at birth due to failure of
excitation-contraction coupling in skeletal muscle that results
from a mutation in the gene for the voltage-sensitive calcium
channel al subunit.  Disruptions have been reported in the
formation of cross-striated myofibrils and in the organization of
the excitation-contraction coupling membrane system in mdg/mdg

mice.  Sections of striated muscle from 18 day embryo wild type
mice or from homozygous or heterozygous mdg mutant mice were
incubated with antibodies directed against the al calcium channel
subunit and examined by indirect immunofluorescence.   The results


11

show that the expression of the al subunit is sharply reduced or
absent in homozygous mdg mutants; however, the spatial
organization of the sarcoplasmic reticulum and myofibrils is

normal, although the intensity of staining is reduced.   T-tubule

organization also is normal, although the intensity of staining is

reduced.  These results suggest that the al calcium channel
subunit is not required for the assembly of the

excitation-coupling membrane system,  and that the absence of the

CX~ subunit may indirectly inhibit myotube maturation.

In cultured rat myotubes,  transverse tubule membranes differ in
molecular composition from plasma membranes. The precursors of
transverse tubules appear to be intracellular vesicles or tubules

that first appear in myoblasts.  Intracellular tubular membranes
with markers for both transverse tubules and sarcoplasmic
reticulum are associated with the 2 lines of immature myofibrils

in newly formed myotubes.

Catabolite gene activator protein (CAP) in the presence of
stimulates transcription from several operons in a. coli.
CAMP-independent variant, in which alanine- is replaced

CAMP

A

by

(CAP-91), is activat

the wild type CAP.

sine,  which does not

e mechanism of the

regulation, site-specific mutations in serine-46 of HPr were
constructed and then tested for PTS activity.   The results
indicate that imposition of a negative charge in the region of
serine-46 of HPr markedly reduces the activity of HPr as a


12

phosphocarrier.

Since high levels of CAMP are toxic to C.  coli, difficulties have

been experienced in constructing strains that overproduce

adenylate cyclase which catalyzes the synthesis of CAMP. A

plasmid vector suitable for the expression of lethal genes was

constructed and the vector DNA was ligated to genomic DNA for

adenlyate cyclase.  The level of adenylate cyclase in E. coli,

cells transformed with the recombinant plasmid DNA increased about

7,000 fold, corresponding to 30 percent of the total protein. A

relatively simple procedure was devised to purify adenylate

cyclase from extracts after hyperexpression to yield nearly

homogenous protein.