Promoters from chlorella virus genes providing for expression of genes in prokaryotic and eukaryotic hosts

The invention is directed to novel promoters or mutants thereof from Chlorella virus DNA methyltransferase genes. A Chlorella virus gene promoter is operably linked to a first and/or second DNA sequence encoding a gene that is different from the Chlorella virus gene to form an expression cassette. An expression cassette can be introduced into prokaryotic and/or eukaryotic cells and can provide for a high level of expression of the gene encoded by the first and/or second DNA sequence. The invention also provides a method for screening other Chlorella virus genes for promoters that can function to express a heterologous gene in prokaryotic and/or eukaryotic hosts.

BACKGROUND OF THE INVENTION 
Genetic engineering has provided a method to isolate, selectively amplify, 
and express genes encoding desirable traits. These genes are often 
obtained from one organism and transformed into another organism so that 
expression of the gene can be manipulated and maximized. The transfer of 
genes from one organism to another can be used to produce large quantities 
of the gene product as well as to provide the transformed organism with 
improved characteristics or traits. Heterologous genes encoding desireable 
traits can be introduced into a wide variety of prokaryotic and eukaryotic 
hosts. For example, advantageous genes encoding herbicide resistance from 
bacteria can be incorporated into a plant's genome. The bacterial gene can 
then be expressed in the plant cell to confer on the plant cell resistance 
to the herbicide. 
In order for the newly inserted gene to be expressed in a eukaryotic or 
prokaryotic host cell, proper regulatory sequences must be present and in 
the proper location with respect to the gene. These regulatory sequences 
include a promoter region and a 3' nontranslated regulatory region. The 
promoter is a region of DNA sequences located upstream from the coding 
sequence of the gene. The function of a promoter sequence is to allow 
access and position of the transcription enzyme RNA polymerase in the 
vicinity of the transcription initiation site. The promoter DNA sequence 
can contain regulatory sequence that influence the rate and timing of the 
transcription of the gene. For example, insertion of a 21 base pair 
sequence into the 35S cauliflower mosaic virus (CaMV) promoter results in 
a tissue-specific expression in roots (Lam et al., PNAS, 86:7890 (1989).) 
Other sequences, like the TATA box and the CCAAT box in eukaryotic 
promoters, are known to influence the rate and level of gene 
transcription. 
Certain promoters are known to be strong promoters. These promoters direct 
transcription at higher levels than other types of promoters and are 
capable of directing expressions in other types of cells. One of the 
promoters that is a strong promoter in some types of plant cells is the 
35S cauliflower mosaic virus (CaMV) promoter. However, the 35S CaMV 
promoter expression in plants can be variable and is especially so in 
monocotyledonous plants. Thus, strong promoters in one system often are 
not capable of providing for gene expression in a wide variety of both 
prokaryotic and eukaryotic host cells. 
Chlorella viruses are a large group of recently identified viruses that 
infect certain eukaryotic green algae Chlorella. Chlorella viruses can be 
produced in large quantities and can be assayed by plaque formation. 
Chlorella viruses are large (150 to 190 nm) polyhedral plaque forming 
viruses containing greater than 300 kilobases of linear double-stranded 
DNA. The viruses are placed into 16 classes on the basis of plaque size 
antibody reactivity and the nature and abundance of methylated bases in 
their genomic DNA. 
The Chlorella viruses have several unique features. The viruses have enough 
DNA sequence to encode 200 to 300 proteins. It is known that the virus 
contains and encodes 50 structural genes. The viruses also encode several 
DNA methyltransferase genes, DNA restriction endonuclease genes, and DNA 
polymerase genes. The DNA methyltransferase genes and the restriction 
endonuclease genes have been studied as a unique DNA 
restriction-modification system. Because the Chlorella viruses can be 
grown to large quantities and have several unique features, they are good 
candidates for the isolation of factors important in gene regulation. 
Thus, there is a need for identifying and isolating strong promoters that 
are capable of expressing heterologous genes in a wide variety of cell 
types. There is also a need to identify and isolate strong promoters that 
can function in monocotyledonous plants, like wheat or rice. There is also 
a need to identify, isolate and characterize the promoters of the 
Chlorella virus genes including the DNA methyltransferase genes. 
SUMMARY OF THE INVENTION 
The invention is directed to novel promoters or mutants thereof from 
Chlorella virus DNA methyltransferase genes. These novel promoters are 
operably linked to a first DNA sequence encoding a gene that is different 
from the Chlorella virus gene to form an expression cassette. An 
expression cassette can optionally include a 3' nontranslated regulatory 
DNA sequence functional in eukaryotic cells and operably linked to the 
first DNA sequence. The preferred Chlorella virus DNA methyltransferase 
promoters in the expression cassette provide for a high level of 
constitutive gene expression in prokaryotic and eukaryotic cell hosts. 
An expression cassette of the invention can further comprise a second DNA 
sequence encoding a different gene from the first DNA sequence. The second 
DNA sequence is linked to the first DNA sequence and under the control of 
the Chlorella virus promoter. The second DNA sequence preferably encodes a 
reporter gene or a selectable marker gene. 
An expression cassette of the invention is introduced into prokaryotic and 
eukaryotic cells, preferably in a plasmid vector. Plasmid vectors 
including an expression cassette of the invention are used to stably 
transform prokaryotic cells. The preferred transformed prokaryotic species 
include E. coli, phytopathogenic members of the genus Pseudomonas and 
Erwinia, plant associated members of the genux Xanthomonas, and members of 
the genus Agrobacterium. Stably transformed prokaryotic cells are selected 
and are capable of transmitting an expression cassette to progeny cells. 
The transformed progeny cells express the genes encoded by the first 
and/or second DNA sequence under the control of the Chlorella virus 
promoters. 
Eukaryotic cells, preferably plant cells, can be transformed with an 
expression cassette, typically in a binary T.sub.i vector. Transformed 
plant cells can transiently express the genes from the first and/or second 
DNA sequence. Transformed plant cells exhibiting transient gene 
expression, preferably monocotyledonous plant cells, can be converted to 
stably transformed or transgenic plants. Transformed plant cells are 
incubated in the presence of callus induction medium and a selective 
agent. Transformed calli can then be used to generate the transformed or 
transgenic plants. The transgenic plants can be grown and selfed or 
crossed to produce transgenic progeny plants and seeds. The preferred 
transgenic plant of the invention is a monocotyledonous plant having a 
Chlorella virus promoter that provides for a high level of constitutive 
gene expression. 
The invention also provides a method for screening other Chlorella virus 
genes for promoters that can function to express a heterologous gene in 
prokaryotic and/or eukaryotic hosts. Chlorella virus genes and 5' flanking 
DNA sequences are isolated, sequenced, and the coding region of the gene 
identified. Once the Chlorella virus gene and its 5' flanking sequence has 
been identified, a method of the invention involves isolating a DNA 
fragment including about 50 to 2000 nucleotide base pairs of the DNA 
sequence upstream from the coding sequence. An expression cassette is 
formed by combining the DNA fragment with a reporter gene, like 
chloramphenicol acetyltransferase. An expression cassette is used to 
transform prokaryotic and/or eukaryotic hosts and expression of the 
reporter gene is detected. Promoter sequences providing for a high level 
of gene expression in eukaryotic and/or prokaryotic hosts can be 
identified.

DETAILED DESCRIPTION OF THE INVENTION 
This invention is directed to novel promoters from Chlorella virus genes 
that provide for a high level of gene expression in prokaryotic and/or 
eukaryotic cells. A promoter associated with a Chlorella virus gene is 
isolated and operably linked to a DNA sequence encoding a gene different 
from that of the Chlorella virus gene in an expression cassette. An 
expression cassette can be further comprised of a 3' nontranslated 
regulatory DNA sequence functional in eukaryotic cells and/or a second DNA 
sequence encoding a selectable marker gene. An expression cassette can 
also be further comprised of plasmid DNA or a binary T.sub.i vector DNA. 
The vector so formed can be used to deliver an expression cassette to the 
prokaryotic or eukaryotic cells. Transformed bacterial cells, preferably 
Agrobacterium, express high levels of the gene under the control of the 
Chlorella virus gene promoter. Transformed plant cells also express the 
gene or genes controlled by the Chlorella virus promoters at high levels 
and can be used to generate transgenic plants. 
1. Formation of an Expression Cassette 
A. Promoters 
The promoters of the invention are obtained or modified from Chlorella 
virus genes. Chlorella viruses are large (150 to 190 nm) polyhedral 
plaque-forming viruses containing &gt;300 kb of linear double-stranded DNA. 
At least 37 strains of the virus that infect eukaryotic Chlorella-like 
green algae have been isolated and partially characterized. The viruses 
are placed into 16 classes on the bases of plaque size, antibody 
reactivity, and the nature and abundance of methylated bases in their 
genomic DNA. Each of the viral DNAs contains 5-methylcytosine at a 
percentage of cytosine ranging from 0.1% to 47.5%. In addition, 25 of the 
37 viral DNA also contain N.sup.6 methyladenine as a percentage of adenine 
ranging from 1.45% to 37%. The finding of sequence specific methylation 
led to the discovery that these viruses have genes encoding DNA 
methyltransferases and site-specific endonucleases. In addition, these 
viruses contain at least 50 structural genes. 
Some of the Chlorella virus genes, including the 5' flanking regions, have 
been isolated and sequenced. Four of the viral-encoded DNA 
methyltransferase genes have been isolated and sequenced, as reported by 
Shields et al., Virology, 176:16 (1990); Stefan et al., Nucleic Acids 
Res., 19:307 (1991); Narva et al., Nucleic Acids Res., 15:9807 (1987); and 
Zhang et al., Nucleic Acid Res. (submitted for publication) Two DNA 
polymerase genes including 5' flanking regions from Chlorella viruses 
PBCV-1 and NY-2A were isolated and sequenced, as described by Grabherr et 
al., Virology, 188:721 (1992). 
Most genes have regions of DNA sequence that are known promoters and which 
regulate gene expression. Promoter regions are typically found in the 
flanking DNA sequence upstream from the coding sequence in both 
prokaryotic and eukaryotic cells. A promoter sequence provides for 
regulation of transcription of the downstream gene coding sequence and 
typically includes from about 50 to 2000 nucleotide base pairs. Eukaryotic 
promoters usually contain a consensus sequence known as the TATA box 
region, 20-30 base pairs (bp) upstream from the transcription initiation 
site. A second eukaryotic consensus sequence, the CCAAT box region, is 
also found at about 80 bp upstream from the transcription initiation site. 
Prokaryotic promoters typically a have a TATAAT consensus sequence 10 bp 
upstream from the start codon, and a TTGACA 35 bp upstream from the start 
codon. These consensus sequences, as well as other promoter regulatory 
sequences, can be present and function to regulate levels of gene 
expression. For example, a 21 bp element has been identified in the 35S 
cauliflower mosaic virus promoter as providing for root specific gene 
expression, as described by Lam et al., PNAS, 86:7890 (1989). 
Promoter DNA sequences are found upstream and are associated with 
expression of a particular gene. The gene regulated by the promoter 
sequence can be referred to as the native or homologous gene. Some 
isolated promoter DNA sequences can provide for gene expression of 
heterologous gene, that is a gene different from the native or homologous 
gene. Promoter sequences are also known to be strong or weak. A strong 
promoter provides for a high level of gene expression, whereas a weak 
promoter provides for a low level of gene expression. An isolated promoter 
sequence that is a strong promoter for heterologous genes has great 
utility and offers many advantages in the transfer of genes from one 
organism to another by recombinant DNA techniques. 
Some of these advantages include providing a sufficient level of gene 
expression to allow for easy detection and selection of transformed cells 
expressing the heterologous gene. Another advantage is to replace the 
native or homologous promoter with a promoter sequence known to provide 
for a high level of gene expression. A third advantage is to allow for the 
transfer of genes for which a promoter has not been identified. 
The promoters of the invention are promoters or mutants thereof of 
Chlorella virus genes. The promoters can provide for gene expression in 
prokaryotic and/or eukaryotic hosts. The promoters also provide for gene 
expression of heterologous genes. A heterologous gene is a gene which is 
different from the Chlorella virus gene from which the Chlorella virus 
promoter is obtained. The heterologous gene can be obtained from different 
organisms, like a bacteria, plant or mammal. The promoters of the 
invention preferably include from about 50 bp to 2000 bp more preferably 
500 to 2000 bp of DNA sequence and can contain regulatory sequences. The 
promoters of the invention can also include from about 3 to about 150 
nucleotides of the coding sequence of the Chlorella virus gene from which 
the promoter is obtained. 
The preferred promoters of the invention are promoters that substantially 
correspond to promoters for Chlorella virus DNA methyltransferase genes. A 
promoter that substantially corresponds to the promoters for DNA 
methyltransferase genes shares about 85% to 100% DNA sequence homology 
with that promoter while retaining the capacity to provide for expression 
of a heterologous gene. These promoters can preferably function in both 
prokaryotic and eukaryotic cells. The preferred promoters also provide for 
a high level of constitutive expression of heterologous genes. 
The promoter designated AMT-1 is found within about 850 bp upstream from 
the adenyl methyltransferase gene M.CviBIII from Chlorella virus NC-1A. 
The promoter designated AMT-2 is found within about 610 bp from the start 
codon for the adenyl methyltransferase gene M.CviRI from Chlorella virus 
XZ-6E. The promoter sequence designated CMT-1 is found within about 500 bp 
upstream from the start codon for the cytosine methyltransferase gene 
M.CviJI from Chlorella virus IL-3A. The adenyl methyltransferase promoters 
AMT-1 and AMT-2 lack the eukaryotic consensus sequences (TATA and CCAAT) 
and prokaryotic consensus sequences (CTATAAT and TTGACA). 
The sequence of the AMT-1 promoter is shown in Table I (SEQ. ID No: 1). The 
sequence of the AMT-2 promoter is shown in Table II (SEQ. ID No: 2). The 
sequence of the CMT-1 promoter is shown in Table III (SEQ. ID No: 3). 
TABLE I 
__________________________________________________________________________ 
SEQUENCE OF THE AMT-1 PROMOTER 
__________________________________________________________________________ 
ATCAGTAATG 
TGTTAATTGC 
GAACGCTTGT 
AATGGTGAAC 
GAATCCAATT 
CGGAAATGCA 
GTCGACTACA 
ATTATTCTTT 
GACACCTTTG 
TTGACGACGC 
ATGCAAAGTT 
GAATATTGAC 
AATCTCGTAT 
AAATTATTCG 
TTTATGCTGT 
TTCAAATCAT 
ATTGAAGTTC 
ACTGGTTTTA 
GAGTGTCGAA 
AAGTATCATA 
TCAACGATTA 
TAGTATTTAA 
TGACAATACT 
CGCGACTGTC 
ATAGTTTATT 
TTTCAACAAT 
GGAGTCTCGT 
CATCATATCA 
ATTTGACGAA 
TGTTGTTCGT 
ATACAAAATA 
TAACAGATGA 
TTTTATTTGC 
GAATACGAAG 
ATTCTTCTTA 
TGGAGAAGAA 
CCAGTTAATA 
ACAAATCGGA 
AGAAGTTCAT 
ACAGCGTTCA 
AATTATATGA 
CATAGATGAC 
GAAACATTGT 
ACAATTATTA 
CAACGGAGTG 
GTCGTACATA 
CTACAAATGG 
ATTGCCAATA 
GTATTCGCAA 
TGGATACACA 
CCGAGGTTGT 
TGCGAGAAAT 
TTTGTATCAC 
GGTACAATTA 
CCAGGGGGCC 
TTACGCGATA 
TGATTTTATT 
GGCGCCACGA 
TTACGAAGGT 
AAGATTTGGT 
AAAGAAAAAC 
GCAAATGCGA 
TATTAATTTT 
TCGGAATTAA 
TTATAGAAAC 
TTCGGTAGGA 
AATATCGTTT 
TACTGGCAGA 
AAACATTCAT 
AATGGATATT 
ACTCTCATGA 
TGTATTCGCT 
TGTTTTGAAG 
GTAAAGTTGA 
AACTTTTCGT 
TTGTAAATAC 
AAAAAATGTA 
TATGAGTATT 
TGTTGTCGGA 
ATGTCATATC 
AACAATGTTG 
TGTATATATG 
TGTAAACTAA 
AATACACTAT 
ATATTATTTA 
A 
__________________________________________________________________________ 
TABLE II 
__________________________________________________________________________ 
SEQUENCE OF THE AMT-2 PROMOTER 
__________________________________________________________________________ 
GAATTCTACT 
TATATACCAT 
ATCATTTTCC 
ATAACAAATT 
GAAAGTCGAA 
TGATTTACCA 
CGTCCTCCGA 
TTTGTTCTAC 
GCTCTTCAAT 
TTTGTAATAT 
CAATGACATT 
TGAAATACTT 
TCTAACAGTC 
TCTGTTGAAC 
ACTTGTATTT 
TCGTTAATAT 
CACGATTATT 
TAGTGTATCA 
ACTATAATTT 
TTCTCGCTGC 
TTATTGTTAA 
TATCGTTGTC 
TCCGCGAATA 
CCTGTTACGA 
AAATATCATC 
AGGATTATCC 
CGTTCCTTTT 
CAGCAAGTTT 
TTCCGCCTTT 
ACTCGTTCCT 
TTTCAGCAAG 
TTTTTCCGCC 
TTTACTCGTT 
CCTTTTCAGC 
AAGTTTTTCC 
GCCTTTACTC 
GTTCCTTTTC 
GATTTTGCTA 
ACCTTTTTCA 
TTTTCATAAG 
ATTGATTATG 
TTTATAATAT 
TCAGCATATT 
TATGTTCTGT 
TCACATATTA 
ATATATATAA 
ATAAAATGAC 
ACAAAAATGA 
CACAAAAATG 
ACACAAAAAT 
GACACAAAAA 
TGACATAGAA 
TTTACACTTG 
TACACTAGAC 
ACGTGTACAC 
AATATCATAT 
CAACATACGA 
AACAACTTAA 
ATTAAAAAAA 
ATGATTGATT 
TTATAAATT 
__________________________________________________________________________ 
TABLE III 
__________________________________________________________________________ 
SEQUENCE OF THE CMT-1 PROMOTER 
__________________________________________________________________________ 
TGTGATGAAC 
TTGAGTTTTA 
CAAAAATATT 
TCTGGTGGAA 
CTATATATTA 
TAGTCCATCA 
GATAAGAATG 
TCGGATTTGT 
TATCATTCCC 
AAGGGTACAG 
AAGTCCATAT 
GAAATATGTT 
AATCTTGATC 
AAGAATGATT 
GTCATTGTAT 
ATTTAAACCA 
TTTATACAAT 
AAGCGTTGAT 
ATAAGTTTGT 
ATATACGTCA 
TTTCGTTATA 
TCAACAAATG 
TTATCATATT 
ATACGTAAAA 
CTGGCTTAAA 
AAAAAACGAG 
TGTAACTATA 
__________________________________________________________________________ 
Mutants of the Chlorella virus promoters include those in which the DNA 
sequence has been changed including a deletion of nucleotides, insertion 
of nucleotides, and/or substitution of nucleotides. Mutants preferably 
contain a functional fragment of the native Chlorella virus promoter. 
Mutants of the promoters of the Chlorella virus genes can be generated by 
standard methodologies including terminal and internal deletion 
mutagenesis, as described by Mitra et al., Molecular Gen. Genetic, 215:294 
(1989); insertional mutagenesis and site-specific mutagenesis, as 
described by Lam et al., PNAS, 86:7890 (1989). Deletion mutants can be 
generated, usually lacking at least one nucleotide from either the 5' or 
3' terminal ends of the promoter sequence. Internal deletion mutants of 
the promoter can be lacking at least one nucleotide in the internal 
portion of the promoter sequence. The insertional mutants can be generated 
by adding DNA sequence either synthesized by automated methods or 
subcloned from another genome and inserted into an intact promoter or a 5' 
or 3' deletion mutant. A mutant promoter sequence can also be generated by 
site-specific mutagenesis by insertion of a synthesized oligonucleotide 
sequence having site-specific mutations. The synthetic oligonucleotide 
sequence can be inserted into the promoter at a specific restriction 
endonuclease site. Mutant promoter sequences can be tested for the ability 
to provide for expression of a heterologous gene by the method of the 
invention. 
Deletion mutants removing up to about 300 bp from the 5' terminus of the 
AMT-1 promoter are the preferred mutants for providing an AMT promoter 
that selectively functions in bacterial cells and does not function in 
tobacco cells. Other deletion mutants generated from the 3' end or 
internally, as well as insertion mutants, can provide for selective 
expression in plant cells and tissue specific expression in plants. 
Promoters from Chlorella virus genes are identified usually in the AT-rich 
5' flanking regions of the coding sequence of Chlorella virus genes, 
preferably DNA methyltransferase genes. Once identified as a putative 
promoter sequence, the promoter sequence is subcloned by digesting the 
cloned Chlorella gene sequence with restriction endonucleases to isolate 
the promoter region and optionally about 3 to 150 nucleotides of the 
Chlorella virus gene coding sequence. Mutants of the promoter sequence can 
be generated as described previously. The digested promoter or mutant 
promoter sequence is operably linked to a promoterless heterologous gene, 
such as the chloramphenicol acetyltransferase gene in a plasmid, and 
ligated with T.sub.4 DNA ligase. The resulting plasmids are introduced 
into prokaryotic and/or eukaryotic cells. Transformed bacteria or plant 
cells can be selected, preferably for antibiotic resistance. The selected 
bacterial or plant cells are then assayed for expression of the 
heterologous gene under the control of the Chlorella virus promoter. 
Other promoters are present in the Chlorella virus genomes and can be 
identified and tested for the ability to provide for expression of 
heterologous genes in both eukaryotic and prokaryotic cells in a method 
provided for in the invention. For example, DNA polymerase genes from 
Chlorella virus PBCV-1 and NY-2A have been isolated and sequenced, as 
described by Grabherr et al., cited supra. The cloned gene sequence 
includes about 160 to 170 nucleotides upstream that are AT rich and could 
function as a promoter. This 5' region of the Chlorella virus DNA 
polymerase genes can be isolated by restriction endonuclease digestion and 
subcloned into a ColEI plasmid or binary Ti vector upstream from a 
promoterless reporter gene, such as chloramphenicol acetyltransferase 
gene. Subcloning this putative promoter region immediately upstream from a 
promoterless reporter gene such as chloramphenicol acetyltransferase 
genes, provides a method for testing whether the DNA sequence having 
potential promoter activity can function to express a heterologous gene in 
a prokaryotic and/or eukaryotic cell. The ColEI plasmids or binary T.sub.i 
vectors can be introduced into prokaryotic or eukaryotic cells and 
transformed cells can be assayed for expression of the reporter gene, such 
as chloramphenicol acetyltransferase gene. Thus, other putative Chlorella 
virus gene promoters can be identified, isolated and tested for the 
ability to provide for gene expression in both prokaryotic and/or 
eukaryotic cells. 
B. DNA Sequence Encoding a Heterologous Gene Different from the Chlorella 
Virus Gene 
An expression cassette is formed by combining one or more DNA sequences 
encoding desired genes with transcriptional and translational regulatory 
DNA sequences that provide for gene expression in a particular cell type. 
The DNA sequences are combined so that the transcriptional and 
translational control regions are operably linked to the DNA coding 
sequence and can function to provide for gene expression. When one or more 
DNA sequences coding for genes are combined with a different promoter 
sequence than the native sequence, the resulting product can be called a 
"DNA construct" or a "fused gene". 
An expression cassette of the invention is formed by combining a promoter 
or mutant thereof from a Chlorella virus gene with one or more DNA 
sequences that encode a gene different from the Chlorella virus gene. The 
DNA sequence preferably encodes a gene that provides a desired 
characteristic in a prokaryotic or eukaryotic host. The gene can be 
another Chlorella virus gene, from a prokaryotic or a eukaryotic organism. 
The gene can also serve as a selectable marker or reporter gene. The 
desired gene will be selected depending upon whether expression is desired 
in prokaryotic cells, eukaryotic cells, or both, and depending on the 
desired trait. 
Specific examples of the types of genes that can form an expression 
cassette of the invention include plant cell genes of economic importance, 
like growth promoter genes, disease resistance genes, frost and drought 
tolerance genes, herbicide and insecticide resistance genes, and the like. 
Desired bacterial genes include genes of economic importance for 
phyllosphere or rhizosphere bacteria associated with plants such as genes 
encoding oxalic acid degradation, genes encoding chitinase, genes encoding 
lignin degradation, and genes encoding biosynthesis of phenazine effective 
to control wheat root disease. In phytopathogenic bacteria, it is 
desirable to include genes that encode antisense messages for toxin genes 
that are associated with plant disease such as tagetoxin. Other genes 
encoding antisense messages can be selected from those described in Plant 
Pathogenic Bacteria, Klement et al. editors, Proceedings of the 7th 
International conference on Plant pathogenic Bacteria, Part A/Part B 
(1990). Desired eukaryotic genes include genes for peptide hormones, 
growth factors, cytokines, and the like. The DNA sequence can also encode 
a selectable marker gene or a reporter gene that provides for selection of 
the transformed prokaryotic or eukaryotic cells. 
The preferred first DNA sequence of the expression cassette encodes 
resistance to a herbicide such as the 5-enol-pyruvyl-phosphoshikimate gene 
that encodes resistance to the herbicide glyphosate. The preferred first 
DNA sequence for use in transformation of phytopathogenic bacteria encodes 
an antisense expression of pathogenic determinate, such as a toxin. 
An expression cassette of the invention can also include a second DNA 
sequence linked to the first DNA sequence and different from the first DNA 
sequence. The second DNA sequence preferably encodes a selectable marker 
gene or a reporter gene. 
Specific examples of selectable marker genes are neomycin 
phosphotransferase gene, apramycin resistance gene, hygromycin 
.beta.-phosphotransferase gene, dihydrofolate reductase gene, guanine 
phosphoribosyl transferase gene, and the thymidine kinase gene. The second 
DNA sequence can also encode a reporter gene such as chloramphenicol 
acetyltransferase, .beta.-galactosidase, .beta.-glucuronidase, and human 
growth hormone. 
Once the desired gene is selected, its DNA sequence can be isolated from 
the source organism by standard methodologies, as provided in Sambrook et 
al., Molecular Cloning: A Laboratory Manual, Coldspring Harbor 
Laboratories, Cold Spring, N.Y. (1989). Generally, once a desired gene is 
selected, a DNA library is prepared from the source organism. Clones of 
DNA having the desired gene are detected by a variety of methods, 
including hybridization to oligonucleotide probes or detection of 
expression of the gene product. DNA clones containing the gene can be 
subcloned and sequenced. 
Once sequenced, the portion of the DNA sequence encoding the coding 
sequence for the gene and lacking a functional native promoter can be 
combined with Chlorella virus promoter in a ColEI plasmid or binary 
T.sub.i vector. Briefly, the DNA sequence encoding the gene and the ColEI 
plasmid or the binary T.sub.i vector are digested with one or more 
restriction endonucleases and then ligated together with T.sub.4 DNA 
ligase. Resulting plasmids incorporating the desired gene lacking all or 
part of the native promoter are selected and amplified, usually in a 
bacterial host. The plasmid carrying the desired gene can then have a 
Chlorella virus promoter sequence inserted upstream so that the promoter 
is operably linked to DNA coding sequence, typically at a different 
restriction endonuclease site. Alternatively, the promoter sequence and 
the DNA sequence encoding the gene can be ligated together first and then 
inserted into a plasmid for selection and amplification. 
C. 3' Nontranslated Regulatory Region 
Optionally, when necessary for efficient gene expression, the expression 
cassette can include a 3' nontranslated regulatory DNA sequence. The 3' 
nontranslated regulatory DNA sequence preferably includes from about 3 to 
1000 nucleotide base pairs (bp) and contains transcription and/or 
translation termination sequences. The 3' nontranslated regions can be 
obtained from the flanking regions of genes from bacterial, plant, or 
other eukaryotic cells. For transcription efficiency and termination of a 
first DNA sequence encoding a prokaryotic gene, the 3' flanking sequences 
can include a transcription termination sequence. For transcription 
efficiency and termination of a first DNA sequence encoding a eukaryotic 
gene, the 3' flanking sequence has a polyadenylation sequence that 
functions to add a polyA tail to the messenger RNA. The 3' nontranslated 
regions are operably linked to the first and/or second DNA sequence to 
provide for gene expression in prokaryotic and eukaryotic cells by 
standard methodologies, as described in Sambrook et al., cited supra. 
Specific examples of the 3' nontranslated regulatory DNA sequences 
functional in eukaryotic cells include about 500 bp of 3' flanking DNA 
sequence of the pea ribulose biphosphate carboxylase small subunit E9 
gene, 3' flanking DNA sequence of the octopine synthase gene, the 3' 
flanking DNA sequence of the nopaline synthase gene, and SV40 
polyadenylation and transcription termination sequences. Especially 
preferred are the 3' nontranslated regulatory DNA sequences that function 
in plant cells such as the 3' flanking DNA sequence from the octopine 
synthase or nopaline synthase genes. 
The 3' nontranslated DNA regulatory regions are often already present in 
plasmid vectors used for selection amplification and transformation of 
prokaryotic and eukaryotic cells. Typically, the desired first and/or 
second DNA sequence encoding the desired genes are inserted immediately 
upstream from 3' nontranslated DNA regulatory sequence so that the DNA 
sequences are operably linked together. Alternatively, the 3' 
nontranslated DNA regulatory regions known to be functional in prokaryotic 
or eukaryotic cells can be isolated from a cloned gene sequence by 
restriction endonuclease digestion. Once isolated, the 3' flanking region 
DNA sequence can be inserted downstream from the first or second DNA 
sequence by standard subcloning methods, as described by Sambrook et al., 
cited supra. 
2. Formation of a Vector Containing the Expression Cassette 
Vectors include additional DNA sequences that provide for easy selection, 
amplification, and transformation of the expression cassette in 
prokaryotic and eukaryotic cells. The additional DNA sequences include 
origins of replication to provide for autonomous replication of the 
vector, selectable marker genes preferably encoding antibiotic resistance, 
unique multiple cloning sites providing for multiple sites to insert the 
expression cassette, and sequences that enhance transformation of 
prokaryotic and eukaryotic cells. The preferred vectors of the invention 
are plasmid vectors. The especially preferred vectors are the ColEI 
plasmid vector or the binary T.sub.i vector pGA582. 
ColEI plasmid vectors, such as pUC18 and pUC19, have been previously 
characterized by Yanisch-Perron et al., Gene, 33:103 (1985) and are 
available from Stratagene or New England BioLabs. A ColEI plasmid vector 
is a 2.7 kbp plasmid and contains an origin of replication that provides 
for autonomous replication in both prokaryotic and eukaryotic cells. A 
ColEI plasmid also contains selectable marker genes encoding antibiotic 
resistance. A ColEI plasmid contains multiple cloning sites providing for 
insertion of an expression cassette of the invention. A ColEI plasmid 
carrying an expression cassette of the invention can be used to transform 
both prokaryotic and eukaryotic cells. 
The binary T.sub.i vector pGA582 has been previously characterized by 
An,Methods in Enzymology, 153:292 (1987) and is available from both Dr. An 
and Dr. Mitra. The binary T.sub.i vector can replicate in prokaryotic 
bacteria, such as E. coli, and in Agrobacterium. The Agrobacterium plasmid 
vectors can be used to transfer the expression cassette to plant cells. 
The binary T.sub.i vectors preferably include the nopaline T-DNA right and 
left borders to provide for efficient plant cell transformation, a 
selectable marker gene, unique multiple cloning sites in the T-border 
regions, the ColEI replication of origin, and a wide host range replicon. 
The binary T.sub.i vectors carrying an expression cassette of the 
invention can be used to transform both prokaryotic and eukaryotic cells, 
but is preferably used to transform plant cells. 
A vector of the invention can also include a vector that can transform 
algal cells, including species of Chlorella. For example, Jarvis et al., 
Current Genetics, 19:317 (1991) have described the use of plasmid pDO432 
derived from the E. coli pUC19 plasmid. This plasmid can contain a 
reporter gene, the nopaline synthase polyadenylation site, and the portion 
of the pUC19 cloning vector (Yanisch-Perron et al., Gene, 33:103 (1985)) 
including the gene for ampicillin resistance and an origin of replication. 
A Chlorella virus promoter fused to a gene encoded by the first DNA 
sequence can be introduced upstream from the reporter gene in a plasmid 
such as pDO432. The plasmid vector can be used to transform Chlorella 
species and other species of green algae. 
A vector of the invention can also be a virus vector. Virus vectors can 
provide for efficient cloning and gene expression in both prokaryotic and 
eukaryotic cells. Specific examples of virus vectors of the invention 
include the M13 phage system, the vaccinnia virus expression system, and 
the baculo virus expression system. 
An expression cassette of the invention can be inserted into or formed 
within a plasmid or virus vector by standard methods. Briefly, the 
Chlorella virus promoter or a mutant thereof can be combined with the 
first DNA sequence encoding a gene having the desired trait to form a 
fused DNA construct. Optionally, a 3' nontranslated DNA regulatory region 
can also be operably linked to the first DNA sequence. The fused DNA 
construct can then be inserted into one of the multiple cloning sites of 
the plasmid vectors by digestion with an appropriate restriction 
endonuclease and ligation with a ligase enzyme. 
Alternatively, each of the DNA sequences in an expression cassette can be 
subcloned into the vector separately. For example, in a binary T.sub.i 
vector having multiple cloning sites, a Chlorella virus promoter sequence 
can be inserted at a unique restriction endonuclease site by digestion 
with that restriction endonuclease followed by ligation. A first DNA 
sequence can then be inserted at a different restriction endonuclease site 
immediately downstream from the Chlorella virus promoter sequence. 
Optionally, and if not already present in the vector, a second DNA 
sequence encoding a selectable marker gene can be inserted at another 
restriction endonuclease site downstream from the first DNA sequence. In 
addition, optionally, if not already present, a 3' nontranslated 
regulatory DNA sequence can be inserted immediately downstream from the 
first or second DNA sequence so that the 3' nontranslated DNA sequence is 
operably linked to the first or second DNA sequence. 
The preferred plasmid of the invention has the characteristics of the ColEI 
plasmid carrying an expression cassette including the AMT-1 promoter fused 
to a promoterless chloramphenicol acetyltransferase gene. The AMT promoter 
provides for a high level of constitutive gene expression in both 
prokaryotic and eukaryotic cells. This ColEI plasmid has been designated 
pAM-15 and has been deposited with the American Type Culture Collection, 
Rockville, Md. E. coli Jm 83 ampR bacteria on Sep. 8, 1992, and given 
Accession No. 69069. 
The especially preferred plasmid has the characteristics of a binary 
T.sub.i vector carrying the AMT promoter fused to a promoterless 
chloramphenicol acetyltransferase gene and also encoding a neomycin 
phosphotransferase gene. The AMT promoter provides a high level of 
constitutive gene expression in plant cells. 
3. Transformation of Prokaryotic and Eukaryotic Cells With An Expression 
Cassette Having a Chlorella Virus Promoter 
An expression cassette is introduced into prokaryotic and/or eukaryotic 
host cells to provide those cells with the capacity to express the desired 
gene encoded by the first and/or second DNA sequence under control of the 
Chlorella virus promoter. Cells containing an expression cassette and 
expressing the gene or genes encoded by the first and/or second DNA 
sequence are referred to as "transformed cells". Gene expression in 
transformed cells can be detected by a variety of methods including 
oligonucleotide probe hybridization to messenger RNA (mRNA), assay for the 
functional activity of the gene product, or detection of the gene product 
by its physical characteristics. Gene expression in transformed cells can 
be transient or stable. Transient gene expression is measured up to about 
72 hours after transformed cells carrying the expression cassette are 
selected and identified. While not in any way meant to limit the 
invention, transient expression indicates that the expression cassette is 
transferred into the cells, the first and/or second DNA sequence is being 
transcribed and translated, and that the gene products are reasonably 
stable in the cell. Stable transformation is exhibited when the cells have 
replicated and the expression cassette can be shown to have been heritably 
transmitted and expressed in progeny cells or organisms. When genes are 
expressed continuously, gene expression is called constitutive. When gene 
expression is turned on and off, gene expression is called inducible. 
An expression cassette of the invention is introduced into prokaryotic and 
eukaryotic cells. The transformed cells can be selected and/or assayed for 
stable and/or transient gene expression. 
An expression cassette of the invention is introduced into prokaryotic host 
cells. The expression cassette is preferably present in a plasmid vector 
such as ColEI plasmid vector or the binary T.sub.i vector. The preferred 
prokaryotic hosts are E. coli, phytopathogenic bacteria including members 
of the genus' Pseudomonas and Erwinia, plant associated nonpathogenic 
bacteria of the genus Xanthomonas, and members of the genus Agrobacterium. 
Especially preferred bacterial hosts are members of the genus 
Agrobacterium that can then be used to transform plant cells. 
An expression cassette can be introduced into bacterial hosts by standard 
methods, preferably by the calcium co-precipitation method. Transformed 
cells carrying an expression cassette in a plasmid can be first selected 
for antibiotic resistance encoded by a selectable marker gene present in 
the plasmid vector. Colonies resistant to the antibiotic and/or 
transformed cells are analyzed for expression of the gene encoded by the 
first and/or second DNA sequence by either mRNA hybridization, assay of 
gene product activity, or detection of the presence of the gene product. 
Typically, prokaryotic host cells are stably transformed with an 
expression cassette and can heritably transmit the expression to the 
progeny cells. 
In a preferred version, phytopathogenic species of the genus Pseudomonas 
and Erwinia are transformed with ColEI plasmid vector carrying the AMT-1 
promoter fused to a promoterless gene such as the chloramphenicol 
acetyltransferase gene. The transformed bacterial cells can be selected 
for antibiotic resistance and then assayed for gene expression of the 
chloramphenicol acetyltransferase gene. The chloramphenicol 
acetyltransferase gene can serve as the first or second DNA sequence. When 
the chloramphenicol acetyltransferase (CAT) gene serves as the second DNA 
sequence, CAT activity can be monitored as a reporter gene for the 
selection of transformed cells carrying the Chlorella virus promoters 
fused to a first DNA sequence encoding the desired gene. The preferred AMT 
promoter provides for a high level of constitutive gene expression in both 
E. coli and phytopathogenic bacteria. 
In the especially preferred version, a binary T.sub.i vector carrying the 
AMT-1 promoter fused to CAT DNA constructs is introduced into 
Agrobacterium species from E. coli. The binary T.sub.i vector is 
transferred into an Agrobacterium species having a helper T.sub.i plasmid. 
The host Agrobacterium strain and helper T.sub.i plasmid can be selected 
depending on the plant species to be transformed. The binary vectors can 
be transferred to Agrobacterium species by a triparental mating method or 
direct DNA transfer method as described by An et al., Methods in 
Enzymology, 153:292 (1987). 
Plasmid vectors carrying an expression cassette comprising a mutant of the 
Chlorella virus promoter fused to a first DNA sequence can also be 
introduced into prokaryotic or eukaryotic host cells. Transformed cells 
can be initially selected, preferably by resistance to an antibiotic. 
Antibiotic resistant cells or transformed cells are examined for 
expression of the gene encoded by the first and/or second DNA sequence. 
The mutant Chlorella virus promoter sequences can provide for selective 
expression in prokaryotic or eukaryotic cells and can also provide for 
tissue-specific expression. The preferred mutants are deletion mutants of 
the Chlorella virus AMT-1 promoters. 
An expression cassette of the invention can be introduced into eukaryotic 
cells, preferably plant cells. An expression cassette preferably in a 
ColEI or binary T.sub.i plasmid vector be introduced into eukaryotic cells 
by direct DNA transfer techniques such as protoplasting, electroporation, 
biolistic transformation, and Agrobacterium-mediated transformation. The 
preferred plant cells to be transformed include corn, rice, wheat, 
tobacco, and Arabidopsis cells. The especially preferred cells are from 
monocotyledonous plants such as rice, corn or wheat. 
Other types of eukaryotic cells can also be transformed by vectors 
including an expression cassette of the invention. Eukaryotic cells such 
as yeast can be transformed using the calcium phosphate coprecipitation 
method. A Chlorella virus promoter of the invention can also be inserted 
into other vectors useful to transform eukaryotic cells. For example, a 
Chlorella virus promoter can be inserted into viral expression systems 
such as vaccinnia virus. Once inserted into these expression systems, they 
can be combined with a DNA sequence encoding the desired gene to provide 
for gene expression in eukaryotic cells. 
In a preferred version, a binary T.sub.i vector or a ColEI plasmid carrying 
the AMT-1 promoter fused to a reporter gene, such as a promoterless 
chloramphenicol acetyltransferase gene, is introduced into wheat, corn, 
rice and tobacco cells by electroporation. Transformed plant cells are 
examined for transient expression of reporter gene-chloramphenicol 
acetyltransferase gene by detection of the enzymatic activity within about 
72 hours of transformation. The chloramphenicol acetyltransferase gene can 
be encoded by the first or second DNA sequence. When encoded by the second 
DNA sequence, the chloramphenicol acetyltransferase gene serves as a 
reporter gene that provides for identification of plant cells transformed 
with the first DNA sequence encoding the desired trait and expressed under 
the control of the Chlorella virus promoter. The preferred AMT promoter 
provides for a high level of constitutive gene expression in plant cells. 
Transformed plant cells exhibiting transient gene expression under control 
of a Chlorella virus promoter or a mutant thereof can be cultured to 
generate stable transformants. Stable transformants can be generated by 
growth on medium that induces callus formation. Alternatively, confirmed 
transformants of haploid cell lines, such the wheat cell line Pavon 64, 
can be doubled with colchicine or crossed with viable pollen to generate 
seeds. 
Plants cells, calli or organs can also be transformed by Agrobacterium 
species carrying a binary T.sub.i vector including an expression cassette 
of the invention. The transformed plant cells, preferably tobacco or 
Arabidopsis, can form calli after plant cell growth on calli induction 
medium over a period of about 2 to 4 weeks. Plant organs, such as leaf, 
stem, hypococotyls, and cotyledons, can also be transformed by 
co-cultivation with Agrobacterium species carrying binary T.sub.i vectors. 
Transformed plant organs can also be induced to form calli. The 
transformed calli are preferably grown in the presence of selective 
agents, such as antibiotics. After the transformed calli are formed and 
selected, the expression of the genes under control of a Chlorella virus 
promoter encoded by the first and/or second DNA sequence is detected. The 
transformed calli exhibit stable transformation. While not in any way 
meant to limit the invention, it is believed that replication and cell 
division in the formation of calli from the transformed plant cells or 
organ tissues, indicates that the expression cassette is stably integrated 
into the plant cell genome and is replicated and transmitted to progeny 
cells. 
4. Formation of Transgenic Plants 
The transformed plant calli exhibiting stable expression of the genes 
encoded by the first and/or second DNA sequence under the control of a 
Chlorella virus promoter or a mutant thereof can be used to generate 
transgenic plants. Methods of generating transgenic plants from calli are 
described in Plant Molecular Biology Manual, Kluwer Publishing (1988). 
Briefly, transformed plant cells are grown on callus or shoot induction 
medium containing an a selective agent, typically an antibiotic until 
calli are formed, generally about 2-4 weeks. Transformed calli can be 
induced to form shoots in the presence of cytokinin in the medium. 
Transgenic plants can be regenerated after 4-6 weeks of incubation from 
shoot cultures. 
Transgenic plants can be crossed with other transgenic and/or nontransgenic 
plants. The next (F.sub.1) generation of plants can be examined for 
heritable transmission of an expression cassette of the invention. 
Transgenic progeny plants to which an expression cassette has been 
inheritably transmitted can provide transgenic seeds containing a 
heterologous gene under the control of a Chlorella virus promoter. 
5. Method of Identifying Whether a Promoter or Mutant Thereof a Chlorella 
Virus Gene Can Function to Express a Heterologous Reporter Gene 
The invention also provides a method for identifying whether a putative 
Chlorella virus gene promoter sequence or a mutant Chlorella virus gene 
promoter sequence can provide for gene expression of a heterologous gene. 
A heterologous gene is a gene different from the original or native 
Chlorella virus gene. The heterologous gene is preferably a promoterless 
reporter gene such as chloramphenicol acetyltransferase, 
.beta.-galactosidase, .beta.-glucuronidase, or human growth hormone. 
In the method of the invention, a DNA fragment is isolated from a Chlorella 
virus gene. Once a Chlorella virus gene sequence has been cloned and 
identified, about 50 to 2000 nucleotide base pairs upstream from the 5' 
end of the coding sequence of the Chlorella virus gene can be isolated by 
restriction endonuclease digestion. This DNA fragment can then be operably 
linked to a heterologous gene to form an expression cassette. 
An expression cassette can be formed by standard methodologies as described 
previously. Briefly, the DNA fragment encoding the putative promoter 
region or the mutant promoter region can be combined with the heterologous 
gene in vector such as a plasmid as follows. A colEI plasmid having 
multiple cloning sites can be digested with a restriction endonuclease 
specific for one of the sites. A heterologous gene such as the 
promoterless chloramphenicol acetyltransferase (CAT) is also digested with 
the same restriction endonuclease. The plasmid digest and the CAT gene are 
mixed and ligated with a ligase enzyme. The colEI plasmids carrying the 
CAT gene are selected and amplified. These plasmids are then digested with 
a restriction endonuclease that cleave at a multiple cloning site upstream 
from the newly inserted CAT gene. The DNA fragment containing putative 
Chlorella virus or mutant Chlorella virus promoter is mixed with the 
digested plasmid and ligated with ligase. Plasmids having the putative or 
mutant Chlorella virus promoter operably linked to the heterologous 
reporter gene, such as the gene encoding chloramphenicol 
acetyltransferase, are selected. 
An expression cassette so formed is transformed into prokaryotic or 
eukaryotic host cells by standard methods. The standard methods include 
calcium coprecipitation, electroporation, biolistic transformation, and 
the like. The preferred transformed host cell is E. coli. 
The expression of the heterologous gene can be detected by standard 
methods. Those methods include radiolabelled oligonucleotide probe 
hybridization to host cell mRNA, physical detection of the gene product 
and/or detection of the functional activity of the gene product. The 
detection of gene expression indicates that the putative or mutant 
promoter Chlorella virus gene sequence can function to provide expression 
of a heterologous gene. 
In a preferred version, a putative or mutant promoter DNA sequence from a 
Chlorella virus DNA methyltransferase gene is combined with a promoterless 
reporter gene, such as the chloramphenicol acetyltransferase gene, in a 
colEI plasmid. The colEI plasmid is introduced into an E. coli host and 
the expression of the chloramphenicol acetyltransferase gene is evaluated. 
The expression of the chloramphenicol acetyltransferase gene is monitored 
by detecting the enzyme activity of chloramphenicol acetyl transferase in 
transformed cells. 
EXAMPLE 1 
Construction of a Vector Containing An Expression Cassette Including 
Promoters from Chlorella Virus DNA Methyltransferase Genes Fused with a 
Chloramphenicol Acetyltransferase Gene (CAT) 
Promoters 
A promoter of the invention is obtained or modified from a Chlorella virus 
gene. Chlorella viruses are large (150 to 190 nm) polyhedral 
plaque-forming viruses containing &gt;300 kb of linear double-stranded DNA. 
At least 37 strains of the virus that infect eukaryotic Chlorella-like 
green algae have been isolated and partially characterized, as described 
in Van Etten et al., Microb. Rev., 55:586 (1991). The viruses are placed 
into 16 classes on the bases of plaque size, antibody reactivity, and the 
nature and abundance of methylated bases in their genomic DNA. Each of the 
viral DNAs contains 5-methylcytosine at a percentage of cytosine ranging 
from 0.1% to 47.5%. In addition, 25 of the 37 viral DNA also contain 
N.sup.6 methyladenine as a percentage of adenine ranging from 1.45% to 
37%. The finding of sequence specific methylation led to the discovery 
that these viruses have genes encoding DNA methyltransferases and 
site-specific endonucleases. In addition, these viruses contain at least 
50 structural genes. 
Some of the Chlorella virus genes, including the 5' flanking regions, have 
been isolated and sequenced. Four of the viral-encoded DNA 
methyltransferase genes have been isolated and sequenced, as reported by 
Shields et al., Virology, 176:16 (1990); Stefan et al., Nucleic Acids 
Res., 19:307 (1991); Narva et al., Nucleic Acids Res., 15:9807 (1987); and 
Zhang et al., Nucleic Acid Research (submitted for publication). Two DNA 
polymerase genes including 5' flanking regions from Chlorella viruses 
PBCV-1 and NY-2A were isolated and sequenced, as described by Grabherr et 
al., Virology, 188:721 (1992). 
The gene encoding an adenyl methyltransferase gene including 5' and 3' 
flanking regions from Chlorella virus NC-1A was cloned into plasmid pUC8 
and expressed in E. coli as described by Narva et al., cited supra. 
Briefly, virus NC-1A DNA was purified and a library of Sau3A partial 
digestion products of the NC-1A DNA was prepared in E. coli plasmid pUC8 
by standard procedures, as described in Mariatis et al., Molecular 
Cloning: A Laboratory Manual, Coldspring Harbor, N.Y. (1982). A clone 
containing the M.CviBIII gene, plasmid NC-1A.14.8 was selected from NC-1A 
DNA library by a procedure originally suggested by Mann et al., Gene, 
3:97-112 (1978) to clone bacterial methyltransferase genes. The 
restriction endonuclease SalI (GTCGAC) and TaqI (TCGA) are inhibited by 
the presence of 6-methyl-deoxyadenine in their recognition sequence. Since 
TCGA sequences are methylated by M.CviBIII, recombinant plasmids 
containing an M.CviBIII gene functional in E. coli are resistant to SalI 
and TaqI. These plasmids were selected by treating the plasmid library 
with SalI before transforming E. coli. 
Several transformants of two SalI resistant clones were obtained when 
10.sup.5 transforming units were digested before transformation of E. coli 
LE392. One clone, pNC-1A.14.8, contained a 2.1 kilobase pair (kbp) insert 
of NC-1A DNA and was resistant to SalI and TaqI when propagated in E. 
coli. A restriction endonuclease map of the 2.1 kbp fragment indicated a 
single NdeI site about 1.2 kbp from one end and no internal BamHI, BglIV, 
SstI, and HindIII sites. When Southern blots of NC-1A DNA digested with 
these restriction endonucleases were probed with nick-translated 
pNC-1A.14.8, two NdeI fragments and single BamHI, BglII, SstI, and HindIII 
fragments were identified as expected. Two fragments were also present in 
double digests of NdeI plus one of each of the other four restriction 
endonucleases. No hybridization to dot blots of host Chlorella NC64A DNA 
was observed. These results indicate that NC-1A Chlorella virus contains a 
single copy of the M.CviBIII gene and that the host Chlorella does not 
contain the gene. 
The DNA sequence of the entire 2.1 kbp fragment containing the M.CviBIII 
gene was determined by dideoxynucleotide chain termination sequencing 
method and reported by Narva et al, cited supra. A single open reading 
frame (ORF) of 1131 base paris (bp) was identified within the functional 
domain predicted by Tn5 mutagenesis which could encode a polypeptide of 
377 amino acids with a molecular weight of 42,828. It was assumed that the 
ATG codon was the initiation codon for M.CviBIII for two reasons: (i) an 
ochre (TAA) codon immediately precedes ATG, and (ii) fusion of the lacZ 
amino terminus at this site results in overproduction of M.CviBIII. 
The Tn5 mutagenesis of recombinant plasmids, including pNC-1A.14.8, was 
performed as described by deBruijn et al., Gene, 27:131 (1984). Sites of 
Tn5 insertion within the M.CviBIII gene were mapped with HindIII and 
HindIII plus EcoRI. Insertional inactivation of the M.CviBIII gene was 
determined by testing the TaqI sensitivity of individual plasmids 
containing unique Tn5 elements. 
A 851 bp putative promoter region present in the 5' flanking region of the 
M.CviBIII adenyl methyltransferase gene was subcloned into either ColEI 
plasmids or a binary T.sub.i vector. The plasmid NC-1A.14.8 containing the 
2.1 kbp fragment was digested with HindIII and DraI to excise the 851 bp 
region. The promoter region so isolated was designated the AMT-1 promoter 
and was placed upstream of a promoterless chloramphenicol 
acetyltransferase (CAT) coding sequence in the ColEI plasmid or the binary 
T.sub.i vector by standard methods, as described in Sambrook et al., cited 
supra. The sequence of the 851 bp promoter designated AMT-1 is shown in 
Table I (SEQ. ID No: 1). 
A second promoter region was obtained from digestion of the pNC-1A.14.8 
with HindIII and XbaI. The second promoter fragment also contained the 
coding sequence for 53 amino terminal amino acids of the M.CviBIII 
methyltransferase protein. The second promoter construct was also 
subcloned upstream from the promoterless CAT coding sequence in ColEI 
plasmids or the binary T.sub.i vectors. 
The adenine methyltransferase gene M.CviRI from Chlorella virus XZ-6E was 
cloned and sequenced as described by Stefan et al., cited supra. DNA 
libraries of EcoRI digestion products of Chlorella virus XZ-6E DNA were 
prepared in plasmid pBR322 and transformed in E. coli by standard methods 
(Maniatis et al., 1982). 
Potential clones containing the M.CviRI and M.CviRII genes were selected 
from the XZ-6E DNA library. The restriction endonuclease PstI (CTGCAG) is 
inhibited by 6 methyladenine in the TGCA portion of its recognition 
sequence. Since M.CviRI methylates adenine in TGCA sequences, recombinant 
plasmids containing a M.CviRI gene which is expressed in E. coli are 
resistant to PstI. Likewise, clones containing M.CviRII were screened by 
treating the library with RsaI; RsaI cleaves GTAC but not GT.sup.n AC 
sequences. Resistant plasmids were selected by treating the XZ-6E plasmid 
library with either PstI or RsaI before transforming E. coli. 
About 100 transformants were obtained after PstI digestion of the partial 
EcoRI XZ-6E library. Plasmid DNA was isolated from 30 individual colonies 
and tested for resistant to PstI. One clone, named pXZ-6E5.9, contained a 
5.9 kb insert of XZ-6E DNA and was resistant to PstI and CviRI. Plasmid 
pXZ-6E5.9 was sensitive to three other restriction endonucleases, RsaI, 
TaqI, and HinfI, whereas virus XZ-6E genomic DNA was resistance to each of 
these enzymes. It was concluded that pXZ-6E5.9 contains the M.CviRI gene. 
Subclones of plasmid pXZ-6E5.9 were prepared and tested for 
sensitivity/resistant to PstI and CviRI. These experiments localized the 
M.CviRI gene to a 2.1 kb region at one end of pXZ-6E5.9; this plasmid was 
named pXZ-6E.14. 
The entire viral insert DNA in plasmid pXZ-6E.14 was subcloned and 
sequenced as reported by Stefan, cited supra. Two open reading frames 
(ORF) of 243 (ORF-A) and 1137 bases were identified. These two ORFs could 
code for polypeptides of 81 (predicted molecular weight of 9,504) and 379 
(predicted molecular weight of 42,814) amino acids, respectively. The 
larger ORF is the M.CviRI gene because the amino acid sequence is similar 
to other adenine methyltransferases. 
The putative promoter region of about 610 pb located 5' to the potential 
start codon of the adenyl methyltransferase gene M.CviRI was subcloned 
into either ColEI plasmids or a binary T.sub.i vector. The plasmid 
pXZ-6E.14 containing a 2.1 kbp fragment was digested with restriction 
endonucleases XhoI and BglII to excise the 610 bp region. The promoter 
region so isolated was designated AMT-2 promoter and was placed upstream 
of a promoterless CAT coding sequence in the ColEI plasmid or the binary 
T.sub.i vector by standard methods. The DNA sequence of the AMT-2 promoter 
is shown in Table II (SEQ. ID No: 2). 
The cytosine methyltransferase gene M.CviJI from Chlorella virus IL-3A was 
obtained as described in Shields et al., Virology, 176:16 (1990). Briefly, 
virus IL-3A DNA was purified and DNA libraries of Sau3A and TaqI partial 
digestion products of IL-3A DNA were prepared in E. coli plasmid pUC19 by 
standard methods (Maniatis et al., 1982). 
Potential clones containing the M.CviJI gene were selected from the IL-3A 
DNA library by a procedure originally suggested by Mann et al. cited 
supra. to clone bacterial methyltransferase genes. The restriction 
endonucleases HindIII (AAGCTT) and SstI (GAGCTC) are inhibited by 5 
methylcytosine in the AGCT portion of their recognition sequences. Since 
M.CviJI methylates cytosine in AGCT sequences, recombinant plasmids 
containing a functional M.CviJI gene should be resistant to HindIII and 
SstI. Resistant plasmids were selected by treating the IL-3A plasmid 
library with both HindIII and SstI before transforming E. coli. 
Several transformants were obtained after HindIII and SstI digestion of 
partial Sau3A and TaqI IL-3A libraries. Plasmid DNA from nine colonies, 
all from the Sau3A library, were resistant to CviJI. These colonies had an 
insert DNA of about 10 kb or 7.2 kb. One of the later colonies (named 
pIL-3A.22) was selected for detailed study. As expected, pIL-3A.22 DNA was 
also resistant to AluI and sensitive to other enzymes such as HhaI, Thai 
and HpaII. The pIL-3A.22 contains the M.CviJI gene, however, the insert 
DNA also either contains a second cytosine methyltransferase gene or the 
M.CviJI enzyme is less specific than CviJI. 
Subclones of plasmid pIL-3A.22 were prepared and tested for sensitivity to 
CviJI and HaeII. These experiments localize the M.CviJI gene and 
resistance to HaeII to a 3.7 kb region at the 3' end of pIL-3A.22. The 
M.CviJI gene was further defined by insertional mutagenesis of pIL-3A.22.8 
with transposon Tn5. Plasmids containing Tn5 insertions were assayed 
qualitatively for M.CviJI directed methylization by testing the 
sensitivity of the plasmid DNAs to CviJI as well as HaeII. Independent 
insertions defined a region of 2 kb on pIL-3A.22.8 which could encode 
M.CviJI as well as resistance to HaeII. 
The entire 3731 bp insert DNA from pIL-3A.22.8 was sequenced, as reported 
by Shields et al., cited supra. Three open reading frames of 483, 1101, 
and 486 bp which could code for polypeptides containing 161, 367, and 162 
amino acids, respectively, were identified. The sequence of the 1101 bp 
plus flanking sequences is the M.CviJI because (i) it is the only ORF 
located in the region identified by Tn5 mutagenesis and (ii) it has amino 
acid motifs similar to those of other cytosine methyltransferases. 
The 276 bp putative promoter contained within the 5' flanking region of the 
M.CviJI gene was subcloned upstream from the CAT coding sequence in either 
ColEI plasmids or binary T.sub.i vector. The plasmid pIL-3A.22.8 was 
digested with restriction endonuclease and the promoter region so isolated 
was designated CMT-1 promoter and was subcloned into the ColEI plasmid or 
binary T.sub.i vector by standard methods. The sequence of CMT-1 promoter 
is shown in Table III (SEQ. ID No: 3). 
The putative promoter regions from the DNA methyltransferase genes were 
isolated and subcloned into ColEI or binary T.sub.i vectors by standard 
methods, as described by Sambrook et al. (1989). Briefly, the promoter DNA 
sequences of the methyltransferase genes and the ColEI or binary T.sub.i 
vectors were digested with restriction endonucleases and then ligated with 
T.sub.4 DNA ligase. The resulting plasmids carrying the putative promoter 
regions from the Chlorella virus methyltransferase genes were selected and 
amplified in E. coli MC1000. 
The ColEI plasmids pUC18 and pUC19 are well characterized plasmid as 
described in Yanisch-Perron et al., cited supra, and can be obtained from 
Strategene or New England Biolabs. The chloramphenicol acetyltransferase 
gene is a well known reporter gene and was obtained in plasmid pSVOCAT, as 
described in Gorman et al., Mol. Cell Biol., 2:1044-1051 (1982). The 
chloramphenicol acetyltransferase gene coding sequence lacking native 
promoter sequence was subcloned into the ColEI plasmid and the binary 
T.sub.i vector by standard methods, as described by Sambrook et al., cited 
supra. 
The binary T.sub.i plasmid vector pGA582 was obtained as follows. A binary 
T.sub.i vector for plant transformation and promoter analysis was formed 
as described by An et al., Methods in Enzymology, 153:292 (1987). Binary 
vectors can be manipulated in E. coli and then transferred and maintained 
in Agrobacterium. The plasmid pGA582 is a 13.2 kbp-long binary vector and 
is available from Dr. An or Dr. Mitra. The plasmid has DNA fragments 
(about 700 bp) containing the nopaline T-DNA right border and a fragment 
(600 bp) containing the nopaline T-DNA left border. There are nine unique 
restriction sites in the T-DNA borders for cloning foreign DNA. These are 
HindIII, XbaI, SacI, HbaI, KpnI, ClaI, BglII, ScaI, and EcoRI. The first 
seven sites are clustered in multiple cloning sites. The vector also 
contains the ColEI origin of replication. The pGA582 also carries a wide 
host range replicon and tetracycline resistance gene allowing for stable 
maintenance of the plasmid in E. coli and Agrobacterium. The promoterless 
chloramphenicol acetyltransferase gene was inserted in place of the 2.7 kb 
BglII EcoRI endonuclease fragment containing the ColEI replication and cos 
site. 
The 1.5 kbp DNA sequence inserted in that site contains the CAT gene as 
well as the plant terminator sequence derived from octopine T-DNA 
transcripts. The putative promoter sequences from the Chlorella virus DNA 
methyltransferase genes were inserted upstream from the promoterless CAT 
gene in one of the multiple cloning sites. 
EXAMPLE 2 
Transfer and Expression of Chlorella Virus Promoter-CAT Constructs Into 
Prokaryotic and Host Cells 
The ColEI plasmids or the binary T.sub.i vectors containing either the 
AMT-1, AMT-2, the 5' deleted AMT-1, or the CMT-1 promoters fused to the 
chloramphenicol acetyltransferase (CAT) gene were introduced into 
eukaryotic and prokaryotic host cells, and the expression of 
chloramphenicol acetyltransferase was measured. 
Prokaryotic host cells selected were E. coli, phytopathogenic members of 
the genus Pseudomonas and Erwinia, and Agrobacterium. ColEI plasmids or 
the binary T.sub.i vectors carrying the Chlorella virus promoter-CAT 
constructs were introduced into bacteria by the calcium coprecipitation 
method or by electroporation method. Transformed bacteria carrying 
promoter-CAT constructs were selected in the presence of ampicillin or 
tetracycline. The presence of plasmid DNA containing the Chlorella virus 
promoter-CAT constructs in the selected bacteria was verified by alkaline 
lysis DNA miniprep method, as described in Sambrook et al., cited supra. 
Gene expression in the transformed cells was measured by quantitating the 
activity of the gene product of the chloramphenicol acetyltransferase gene 
after 24-48 hours of culture. The expression pattern of the AMT-CAT 
constructs was, in some cases, compared to the expression of the 35S 
promoter from cauliflower mosaic virus fused to the chloramphenicol 
acetyltransferase gene. 
Chloramphenicol acetyltransferase is assayed in cell extracts in the 
following reaction mixture: 10-20 ml of cell extract, 100 ml of 0.25 M 
Tris-HMl pH=7.8, 10 ml of 4mM acetyl coenzyme A, 50 MCi of [.sup.14 C] 
chloramphenicol (57 mCi/mmol, New England Nuclear). The mixture was 
incubated at 37.degree. C. for 20 minutes. The reaction was stopped by 
adding 1 ml of ethylacetate. The mixture was centrifuged and the 
ethylacetate was removed by evaporation to dryness. The dried pellet was 
dissolved in 30 ml of ethylacetate and run on silica gel thin-layer 
chromatography (TLC). The TLC plate was run in 95% chloroform/5% methanol. 
An X-ray film was exposed to the chromogram overnight and the film 
developed. Radioactive spots migrating with an Rf similar to the standard 
were detected. 
Plasmid pAM-55 was constructed from pGA582 plasmid and contains the 
neomycin phosphotransferase gene and tetracycline resistance gene, as 
described in Mitra and An, Mol. Gen. Genetics, cited supra. The 
promoterless CAT gene was subcloned into BamHI restriction endonuclease 
site. The Chlorella virus promoter sequences were not fused with the 
promoterless CAT gene. This plasmid was used to determine whether the CAT 
structural gene could be driven by any other promoter on the plasmid. E. 
coli MC1000 was transformed with pAM-55 by the calcium coprecipitation 
method. No CAT activity was detected in the transformed cells indicating 
that promoterless CAT gene subcloned into ColEI plasmids was not expressed 
without the Chlorella virus or 35S cauliflower mosaic virus promoters. 
The pAM-15 plasmid was constructed in a ColEI plasmid and contains AMT-CAT 
DNA fused DNA constructs as well as a gene encoding resistance against 
ampicillin. 2 .mu.g of plasmid DNA was used to transform bacterial cells 
by the calcium coprecipitation method. The bacteria transformed were E. 
coli and phytopathogenic bacteria from the genes Erwinia and Bacillus. The 
results are shown in FIG. 1. Lane 1 corresponds to E. coli MC1000; Lane 2 
corresponds to E. coli JN83; Lane 3 corresponds to Bacillus pumulis; Lane 
4 corresponds to Erwinia amylovora; and Lane 5 corresponds to Erwinia 
carotovora; P corresponds to positive CAT control; and N corresponds to 
negative CAT control. 
The results show that the AMT-1 promoter provided for high level of 
constitutive expression of the chloramphenicol acetyltransferase gene in 
E. coli and in some species phytopathogenic species of Erwinia. No 
expression was detected in Bacillus pumulis. 
The pAM-50 plasmid was constructed from pGA582 plasmid, and contained the 
neomycin phosphotransferase gene and tetracycline resistance gene. The 
Chlorella virus AMT promoter fused to CAT construct was also present. 2 
.mu.g of plasmid DNA was used to transform bacterial cells by 
electroporation. The results are shown in FIG. 2. The bacterial cells 
includes Clavibacter michiganense ssp. Nebraskense (Lane 1); Pseudomonas 
syringae pv. syringae (Lane 2); Xanthomonas campestris pv. asclepiadis 
(Lane 3); and Agrobacterium tumefaciens (Lane 4). The positive CAT control 
is shown in Lane 5 and the negative control in Lane 6. 
The results show that the Chlorella virus AMT promoter could be expressed 
in a phytopathogenic bacterial species Pseudomonas syringae pv. syringae, 
as well as Agrobacterium tumefaciens. Thus, the AMT promoter is functional 
in a wide variety of bacterial species, making this promoter useful in 
cloning and expressing heterologous genes in different bacterial species. 
EXAMPLE 3 
Analysis of Promoter Activity of AMT-5' Deletion Mutants 
Promoters have regulatory regions that are essential to regulate levels of 
expression and tissue-specific expression. Mutations of promoter regions 
can significantly change promoter function, including tissue-specific 
promoter expression patterns. Mutant promoter DNA sequences can be 
obtained by known methods of deletion mutagenesis, insertional 
mutagenesis, or site-specific mutagenesis, or combinations thereof as 
described by Lam et al., PNAS, 86:7890 (1989) and Ha et al., PNAS, 85:8017 
(1988). 
Promoter region deletion mutants were generated as described by Ha et al., 
cited supra. Plasmids PUC18 and PUC19 are plasmids that are obtained and 
have the characteristics as described Yanisch-Perron et al., Gene, 33:103 
(1985) (SEQ. ID NO: 4). A synthetic nucleotide (GGTACCTCGAGGCCT) 
containing restriction sites for KpnI, XhoI, StuI was inserted into pUC18 
and pUC19 plasmids in a unique SspI site upstream of the .beta.-lactamase 
gene. The SmaI-DraI fragment containing the AMT-1 promoter region of the 
adenyl methyltransferase gene was then inserted into the HindIII site 
within the LacZ .alpha.-complementation gene of the pUC18 and pUC19 
plasmids. The plasmids were the starting materials for generating 5' and 
3' deletion mutants. 
The resulting plasmids were linearized with HindIII and digested with an 
exonuclease, BAL-31, in solution (600 mM NaCl/12 mM CaCl.sub.2 /12 mM 
MgCl.sub.2 /20 mM Tris-HCl, pH=8.0) at 30.degree. C. The reaction was 
stopped at 1-minute intervals by removing a portion of the mixture and 
adding it into an Eppendorf test tube containing 0.1 ml of a 0.3M sodium 
acetate (pH=7.0), 0.1 ml chloroform, and 0.1 ml phenol. After mixing 
vigorously, the test tube was centrifuged for 2 minutes and the aqueous 
phase was precipitated with 2 vol of ethanol. DNA was dissolved in 10 ml 
of medium-salt buffer (50 mM NaCl/10 mM MgCl.sub.2 /50 mM Tris-HCl, 
pH=8.0), digested with StuI, and self-ligated with T4 DNA ligase. Ligated 
DNA was introduced into E. coli MC1000 and the 5' deletion mutants were 
analyzed by digesting DNA prepared from the ampicillin resistant colonies 
with XhoI and HindIII. The 3' deletion mutants can be generated in a 
similar manner but by placing the synthetic oligonucleotide linker 
downstream from the promoter region. 
Using these methods, three 5' deletion mutants were isolated and 
characterized. The deletion end points were determined by the 
Maxam-Gilbert DNA sequencing method after labelling the deletion end point 
with [.alpha..sup.32 P] dGTP and DNA polymerase large fragment. The 5' 
deletion mutants generated were lacking 304,529 and 753 nucleotide base 
pairs from the 5' end of the AMT-1 promoter. The 5' deletion mutants were 
then fused with the CAT gene and subcloned into ColEI plasmids or the 
T.sub.i binary vectors, as described previously. 
The three 5' deletion mutants of the AMT promoter were used to transform 
competent E. coli cells by the calcium coprecipitation method and analyzed 
for CAT activity. The results are shown in FIG. 3. 
The deletion endpoints of the 5' deletion mutants are shown to the left. 
The deletion end points are measured from the translational start codon. 
The first nucleotide to the left of the start codon is designated -1. The 
AMT promoter mutants lacking 304 nucleotides from the 5' end of the AMT-1 
promoter were functional in E. coli. The AMT promoter mutants lacking 525 
nucleotides (-325) and 753 nucleotides (-101) did not provide for CAT 
expression in E. coli. 
The same 5' deletion mutants of the AMT promoter were also evaluated for 
expression in tobacco calli. Suspension culture tobacco cells were 
transformed with 5' deletion mutants and transformed calli were analyzed 
for CAT activity. The results indicate that only the full length promoter 
(-851) was functional in tobacco calli. 
The analysis of the 5' deletion mutants indicates that for some hosts 
removal of a portion of the AMT-1 promoter sequence did not impair the 
function of the promoter. The expression of one of the 5' deletion mutants 
(-550) in E. coli and not in tobacco cells suggests that mutants of the 
AMT promoters can be generated that are selectively expressed in certain 
host cell types. 
EXAMPLE 4 
Stable and Transient Expression of Chlorella Virus Promoters in Eukaryotic 
and Prokaryotic Cells 
Plasmids containing the AMT-CAT expression cassette were used to transform 
rice, wheat, tobacco, and Arabidopsis cells. Tobacco and Arabidopsis cells 
were cultured for the development of transformed calli. The expression of 
CAT driven by the AMT promoters was compared to the expression of CAT 
driven by the 35S promoter from cauliflower mosaic virus (CaMV). Transient 
expression of CAT was measured after 24-48 hours of culture of the 
transformed cells. Stable expression of CAT was measured in tobacco and 
Arabidopsis calli, Agrobacterium tumefaciens, and E. coli. 
Wheat, rice and tobacco cells were transformed with plasmids containing the 
AMT-CAT expression cassette or the 35S CaMV promoter-CAT expression 
cassette. ColEI plasmids containing the expression cassettes were 
introduced into the plant cells by electroporation. Transient CAT activity 
was measured in the transformed plant cells after 24-48 hours. The results 
are shown in FIG. 4. 
The AMT promoter provided for a high level of transient expression in 
wheat, rice and tobacco cells. The level of expression was higher than 
that seen with the 35S CaMV promoter-CAT constructs. Similar experiments 
were done with the CMT-1 promoter-CAT expression cassette and the results 
also indicated that the CMT-1 promoter could function to provide for gene 
expression in the plant cells (data not shown). 
For analysis of stable transformation, tobacco and Arabidopsis cells were 
transformed with Agrobacterium carrying a binary T.sub.i vector and 
cultured for the development of calli. Agrobacterium tumefaciens was 
transformed using the binary T.sub.i vectors carrying the Chlorella virus 
AMT-promoter fused to CAT or the 35S CaMV promoter fused to CAT. Tobacco 
leaf discs and Arabidopsis cells were co-cultivated with Agrobacterium 
carrying the binary T.sub.i vectors. 
After co-cultivation for two days at 28.degree. C., the bacterial cells 
were washed out and the tobacco leaf discs and Arabidopsis cells were 
grown on callus induction medium containing 200 .mu.g of kanamycin and 250 
.mu.g/ml of carbenicillin. The co-cultivated plant tissues were incubated 
in the dark for callus induction. 
Once transformed tobacco and Arabidopsis calli were formed and selected, 
expression of the AMT-CAT constructs was measured in plant extracts as 
described in Example 2. Plant extracts were obtained by lysis of calli 
with a pestle or by sonication in plant extraction buffer (0.5 molar 
sucrose, 0.1% ascorbic acid, 0.1% cysteine-HCl, 0.1 molar Tris-HCl, 
pH=7.8). The lysate was centrifuged and the supernatant assayed for 
chloramphenicol acetyltransferase. The results are shown in FIG. 4. 
The results in FIG. 4 show that in both transformed tobacco and Arabidopsis 
calli, the AMT promoter chloramphenicol acetyl transferase fused gene was 
strongly expressed. The levels of expression were greater than that of the 
35S cauliflower mosaic virus chloramphenicol acetyltransferase constructs. 
Thus, the AMT promoter provides for high level of transient as well as 
stable gene expression. 
Tobacco and Arabidopsis calli transformed with an expression cassette 
containing Chlorella virus AMT promoter fused to CAT were used to 
regenerate transformed plants. The transformed or transgenic plants, as 
well as the F1 generation of the transformed plants were analyzed for CAT 
expression. Both transformed plants and the F1 generation exhibited strong 
expression of the chloramphenicol acetyltransferase gene indicating that 
the AMT promoter-CAT constructs can be stably transmitted and expressed 
from generation to generation. 
EXAMPLE 5 
Formation of Chlorella Virus AMT-Neomycin Phosphotransferase Gene and 
Hygromycin Resistant Gene Expression Cassettes and Transformation of Wheat 
The AMT promoters were combined with promoterless chloramphenicol 
acetyltransferase (CAT) gene, as described in the previous Examples. The 
expression of this "reporter" gene allowed for a standardized measurement 
of gene expression in a variety of cell types. The AMT promoters were also 
combined with genes known to be plant selectable markers. One of the genes 
is known as neomycin phosphotransferase gene II and is known to encode 
resistance of plant cells to G-418. 
The neomycin phosphotransferase gene is present in the binary T.sub.i 
vector obtained as described in Example 1. A promoterless neomycin 
phosphotransferase gene was obtained by restriction endonuclease digestion 
of the pGA582 plasmid and inserted downstream from the AMT promoter in the 
ColEI plasmid by standard methods. Likewise, a promoterless hygromycin 
resistance gene can be subcloned into the ColEI plasmid downstream from 
the AMT promoter using standard methods provided in Sambrook et al., cited 
supra. 
The haploid wheat cell line, Pavon 64, was transformed with the fused AMT 
promoter-neomycin phosphotransferase gene. Pavon 64 calli were partially 
treated with 0.1% pectolyase Y-23 and 1% cellulase RS. The cells were 
electroporated with 25 .mu.g of plasmid ColEI DNA and 75 .mu.g carrier DNA 
at 400 volts with a capacitance of 800 UFD. The electroporated cells were 
grown on 85D12 regeneration medium without any plant growth regulators as 
described by Liang et al., Crop Sci., 27:336 (1987). 
Transformed cells were assayed for neomycin phosphotransferase activity to 
detect expression of neomycin phosphotransferase gene. Neomycin 
phosphotransferase was assayed by standard methods. Briefly, 10 .mu.l of 
plant extract, 10 .mu.l of assay buffer (40 mM magnesium chloride, 40 mM 
ammonium chloride, 2 mM dithiothreotal, 65 mM Tris-HCl, pH=7.5), 2 .mu.l 
of Kanamycin sulfate and 10 .mu.l of ATP solution (50 .mu.m ATP and 0.1 
mCi [.sup.32 P] ATP, 3000 Ci/mmol) was mixed and incubated for 20 minutes 
at 37.degree. C. The solution was spotted onto 1 cm.sup.2 of Whatman P-81 
phosphocellulose paper and dried under a heat lamp. The spotted Whatman 
paper was washed and then counted in a scintillation counter. 
The results of transformation of the wheat cell line with the AMT-neomycin 
phosphotransferase expression cassettes show that transient gene 
expression was obtained in the embroyides 36 hours after the 
electroporation. Thus, the wheat cells were transformed and could express 
the neomycin phosphotransferase gene under the control of the Chlorella 
virus AMT promoters. 
Stable transformation of wheat plants will be tested by the following 
method. Hygromycin or Kanamycin resistant transgenic wheat plants will be 
doubled with colchicine or crossed with viable pollen to produce seeds as 
described in Liang et al., Crop. Sci., 27:336-339 (1987). Seeds will be 
grown and the F1 plants tested for neomycin phosphotransferase gene 
expression or hygromycin resistance. 
The references are incorporated by reference. The invention has been 
described with reference to various specific and preferred embodiments and 
techniques. However, it should be understood that many variations and 
modifications may be made while remaining within the spirit and scope of 
the invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 851 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Chlorella virus NC- 1A 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AMT-1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATCAGTAATGTGTTAATTGCGAACGCTTGTAATGGTGAACGAATCCAATTCGGAAATGCA60 
GTCGACTACAATTATTCTTTGACACCTTTGTTGACGACGCATGCAAAGTTGAATATTGAC120 
AATCTCGTATAAATTATTCGTTTATGCTGTTTCAAATCATATTGAAGTTCACTGGTTTTA180 
GAGTGTCGAAAAGTATCATATCAACGATTATAGTATTTAATGACAATACTCGCGACTGTC240 
ATAGTTTATTTTTCAACAATGGAGTCTCGTCATCATATCAATTTGACGAATGTTGTTCGT300 
ATACAAAATATAACAGATGATTTTATTTGCGAATACGAAGATTCTTCTTATGGAGAAGAA360 
CCAGTTAATAACAAATCGGAAGAAGTTCATACAGCGTTCAAATTATATGACATAGATGAC420 
GAAACATTGTACAATTATTACAACGGAGTGGTCGTACATACTACAAATGGATTGCCAATA480 
GTATTCGCAATGGATACACACCGAGGTTGTTGCGAGAAATTTTGTATCACGGTACAATTA540 
CCAGGGGGCCTTACGCGATATGATTTTATTGGCGCCACGATTACGAAGGTAAGATTTGGT600 
AAAGAAAAACGCAAATGCGATATTAATTTTTCGGAATTAATTATAGAAACTTCGGTAGGA660 
AATATCGTTTTACTGGCAGAAAACATTCATAATGGATATTACTCTCATGATGTATTCGCT720 
TGTTTTGAAGGTAAAGTTGAAACTTTTCGTTTGTAAATACAAAAAATGTATATGAGTATT780 
TGTTGTCGGAATGTCATATCAACAATGTTGTGTATATATGTGTAAACTAAAATACACTAT840 
ATATTATTTAA851 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 609 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Chlorella virus XZ- 6E 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: AMT-2 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GAATTCTACTTATATACCATATCATTTTCCATAACAAATTGAAAGTCGAATGATTTACCA60 
CGTCCTCCGATTTGTTCTACGCTCTTCAATTTTGTAATATCAATGACATTTGAAATACTT120 
TCTAACAGTCTCTGTTGAACACTTGTATTTTCGTTAATATCACGATTATTTAGTGTATCA180 
ACTATAATTTTTCTCGCTGCTTATTGTTAATATCGTTGTCTCCGCGAATACCTGTTACGA240 
AAATATCATCAGGATTATCCCGTTCCTTTTCAGCAAGTTTTTCCGCCTTTACTCGTTCCT300 
TTTCAGCAAGTTTTTCCGCCTTTACTCGTTCCTTTTCAGCAAGTTTTTCCGCCTTTACTC360 
GTTCCTTTTCGATTTTGCTAACCTTTTTCATTTTCATAAGATTGATTATGTTTATAATAT420 
TCAGCATATTTATGTTCTGTTCACATATTAATATATATAAATAAAATGACACAAAAATGA480 
CACAAAAATGACACAAAAATGACACAAAAATGACATAGAATTTACACTTGTACACTAGAC540 
ACGTGTACACAATATCATATCAACATACGAAACAACTTAAATTAAAAAAAATGATTGATT600 
TTATAAATT609 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 270 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Chlorella virus IL- 3A 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: CMT-1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TGTGATGAACTTGAGTTTTACAAAAATATTTCTGGTGGAACTATATATTATAGTCCATCA60 
GATAAGAATGTCGGATTTGTTATCATTCCCAAGGGTACAGAAGTCCATATGAAATATGTT120 
AATCTTGATCAAGAATGATTGTCATTGTATATTTAAACCATTTATACAATAAGCGTTGAT180 
ATAAGTTTGTATATACGTCATTTCGTTATATCAACAAATGTTATCATATTATACGTAAAA240 
CTGGCTTAAAAAAAAACGAGTGTAACTATA270 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: Multirestriction site oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GGTACCTCGAGGCCT15 
__________________________________________________________________________