A. Mouse Homebox And POU-Domain Genes. A novel mouse homebox gene NKx-1 was cloned an approximately 8 kb was sequenced.
The deduced amino acid sequence of the NKx-1 homeodomain differs from Drosophila NK-1 homeodomain by only 3 of 60 amino acid
residues. Both NKx-1 and NK-1 proteins contain an acididc region before the homeodomain. Northern analysis revealed one
major band of NKx-1 poly A+ RNA in brain and trace bands in testes and spleen. The abundance of NKx-1 poly A+ RNA is highest
in 10 day mouse embryos and progressively decreases thereafter. In situ hybridization with sections of 14-day mouse embryos
revealed NKx-1 RNA in the mesencephalon, myelencephalon, spinal cord, vertebrae, and ribs. Five additional novel mouse homebox
genes were cloned and partially sequenced. A novel mouse POU-domain gene related to Brain-3 POU-domain cDNA was cloned and
about 3 kb was sequenced. The expression of Brain-1, Brain-2, Brain-4 and Skip POU-domain genes in the mouse nervous system
was determined by in situ hybridization as a function of developmental age.
B. Regulation of a Calcium Channel a-1 Subunit Gene. A nucleotide sequence was found in the 5-upstream regulatory region
of a voltage-sensitive calcium channel [alpha]-1 subunit gene that is a powerful activator of an enhancerless chloramphenicol
acetyltransferase reporter gene. A cDNA expression library was screened for recombinants that direct the synthesis of proteins
that bind this nucleotide sequence. Seven kinds of clones were found that encode proteins that bind to oligonucleotides with
appropriate sequence specificity.
C. Promotor And Enhancer Selection. cDNA clones were obtained that encode proteins that specifically bind to oligonucleotide
sequences by a method that selects for gene regulatory sequences. Partial sequences of the cDNA clones were obtained.
Regulation Of Gene Expression
Mouse Homeobox And POU-Domain Genes. Approximately 8 kb of a novel mouse homeobox gene, NKx-1, a homolog of the Drosophila
NK-1 homeobox gene, was sequenced. The amino acid sequences of the NKx-1 and NK-1 homeodomains differ by only 3 of the 60
amino acid residues present. Both proteins also contain an acidic domain. However, most of the other regions of the protein
that have been defined differ markedly. NKx-1 poly A+ RNA was found to be most abundant in 10-day mouse embryos; the abundance
progressively decreases thereafter. Northern analysis of poly A+ RNA from adults revealed 1 major band of NKx-1 poly A+ RNA
in brain and trace bands in RNA from testes or spleen. The NKx-1 gene is expressed in discrete regions of the 14-day old mouse
embryo mesencephalon and myelencephalon and also in spinal cord, vertebrae, and ribs.
A mouse genomic DNA library was screened with oligodeoxynucleotide probes for novel homeobox genes. Seventy-two positive
recombinants were cloned. Thus far five novel homeobox genes have been found. Restriction site analysis of the 72 clones
revealed additional classes of clones that have not yet been identified. In addition, a novel mouse POU-domain gene related
to Brain-3 POU-domain cDNA was cloned and 3 kb was sequenced. Sites of expression of Brain-l, Brain-2, Brain-4, and SKIP
POU-domain genes in the mouse nervous system were determined by in situ hybridization as a function of mouse embryo developmental
age and also were defined in the adult mouse. Hox 4.1 cDNA and genomic DNA were cloned and the complete Hox 4.1 open reading
frame was sequenced.
Regulation of a Calcium Channel [alpha]-l Subunit Gene. The [alpha]-1 subunit of a voltage-sensitive calcium channel previously
was shown to be inducible in NG108-15 cells and the expression of the gene was shown to control the ability of the cells to
form synapses with striated muscle cells. The 5'-upstream regulatory region of the calcium channel gene was cloned and
sequenced. A nucleotide sequence was found that activates an enhancerless chloramphenicol acetyltransferase reporter gene.
A protein was found in NG108-15 nuclei that specifically binds to this sequence. A cDNA expression library in [lambda]gtll
was screened for recombinants that direct the synthesis of proteins that bind to the nucleotide sequence and 35 positive clones
were obtained. Seven kinds of clones were found that encode proteins that bind to oligonucleotides with appropriate sequence
specificity but differ in specificity for double-stranded DNA, or (+) or (-) single-stranded DNA. Further work is needed to
determine whether one or more of the proteins regulate the expression of the Ca2+ gene channel.
Enhancer and Promotor Selection. During the past year further work has been done on the selective amplification of DNA clones
that contain enhancer or promoter nucleotide sequences that activate gene expression. The method is based on the observation
that the synthesis of polyoma virus DNA in mouse cells requires viral enhancer sequences that also are required for the synthesis
of mRNA from polyoma genes. Mouse genomic DNA fragments were ligated to polyoma DNA that lack the enhancer region of the virus.
The E. coli origin of replication and [beta]-lactamase gene also were inserted in the polyoma coat protein gene. Promoters
or enhancers in the mouse genomic DNA inserts that activate plasmid DNA synthesis in mouse cells are able to replicate and
hence are selectively amplified; whereas, plasmids that lack functional enhancer sequences do not replicate. Plasmid DNA
was harvested from mouse cells that had been transfected and incubated for several days. Recovered DNA then was amplified
in E. coli. The selection method is highly effective; some clones were shown to increase in abundance more than 100,000 fold.
Fragments of the recovered DNA inserts were shown to bind nuclear protein and activate the expression of an enhancerless chloramphenicol
acetyltransferase reporter gene. Previously, cDNA clones were obtained that code for proteins that specifically bind to oligonucleotide
sequences that were identified by the oligonucleotide selection method. Partial sequences of some of the cDNA clones were