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The Marshall W. Nirenberg Papers

Title:
Laboratory project: "Regulation of Gene Expression" pdf (301,914 Bytes) transcript of pdf
Laboratory project: "Regulation of Gene Expression"
Number of Image Pages:
3 (301,914 Bytes)
Date:
1993-09 (September 1993)
Creator:
Nirenberg, Marshall W.
Asoh, Sadamitsu
Bethke, Bruce
Hara, Yoshinobu
Nazarali, Adil
Rovescalli, Alessandra
Tan, Dong-Ping
Tsai, Wu-Hong
Guo, Vicky
National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics
Rights:
This item is in the public domain. It may be used without permission.
Exhibit Category:
From Neuroblastoma to Homeobox Genes, 1976-1992
Relation:
Metadata Record Annual Report of the Laboratory of Biochemical Genetics, October 1, 1992 - September 30, 1993 (September 1993)
Box Number: 7
Folder Number: 6
Unique Identifier:
JJBBTK
Accession Number:
2001-013
Document Type:
Reports
Excerpts
Language:
English
Format:
application/pdf
image/tif
Physical Condition:
Good
Series: Series III: Laboratory Administration, [1959]-1993
SubSeries: Annual Reports, 1960-1993
Folder: XXXXXXXXXX, 1988-1993
Transcript:
Project Number: Z01 HL 0155-01 LBG
Period Covered: October 1, 1992 - September 30, 1993
Title of Project: Regulation of Gene Expression
Principal Investigator:
P.I.: Marshall Nirenberg, Chief, LBG, NHLBI
Others: Sadamitsu Asoh, Visiting Associate, LBG, NHLBI
Bruce Bethke, IRTA, LBG, NHLBI
Yoshinobu Hara, Visiting Associate, LBG, NHLBI
Adil Nazarali, Visiting Associate, LBG, NHLBI
Alessandra Rovescalli, Visiting Fellow, LBG, NHLBI
Dong-Ping Tan, Visiting Associate, LBG, NHLBI
Wu-Hong Tsai, Visiting Fellow, LBG, NHLBI
Vicki Guo, Chemist, LBG, NHLBI
Cooperating Units (if any):
Scott Young, LCB, NIMH
Adam Hartman, LCB, NIMH
Yongsok Kim, LMC, NHLBI
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section of Molecular Biology
Institute and Location: NHLBI, NIH, Bethesda, MD
Total Man-Years: 8.5
Professional: 7.5
Other: 1.0
Summary of Work:
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
Project Description
Major Findings
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 obtained.
Metadata Last Modified Date:
2010-11-17
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