The vitellogenin gene family from the nematode, Caenorhabditis elegans is being studied. The genes are expressed abundantly in the intestine of the adult hermaphrodite worm, but are not transcribed in the male, in any of the four larval stages, or inother hermaphrodite tissues. In C. elegans five closely-related genes and one distant member of the gene family encode two distinct classess of vitellogenins. The main objective of this research is to understand the nature of the developmental signals which control transcription of these genes and the detailed mechanisms by which the regulation is effected. By sequencing the promoter regions of the six vitellogenin genes plus those from a related nematode species, two highly conserved heptameric sequences which are likely to be involve in the regulation have been identified. Development of a nematode transformation system to test these hypotheses has just recently been accomplished. A vitellogenin gene promoter has been fused to the structural gene for E. coli beta-glucuronidase and several transgenic worm strains containing the fusion gene integrated in their genomes have been obtained. These lines will be tested for regulated expression. The regions of the promoter required for particular aspects of the regulation will be identified using deletions, linker-scanning mutagenesis, and synthesis of putative regulatory elements. The regulatory roles of the different heptameric element will also be tested in competition experiments: Transformants containing long tandem arrays of the elements will be tested for inappropriate expression. Sequences bound by proteins in crude extracts will be identified by footprinting assays. The proteins that bind to the heptamers will be purified and the genes that encode them will be cloned. Regulatory mutants will be selected and their effects of the binding proteins measured. Mutants that express the vitellogenins in males or that fail to express them in hermaphrodites will be analyzed. Sequence analysis has revealed the presence of potential stem- loop structures at the 5' ends of the vitellogenin mRNAs. Evolutionary evidence strongly suggests that such stems exist. Direct evidence for their existence will be obtained by use of single-and double-strand specific nuclease digestion. Possible functions for the stems will be investigated, and the importance of the stems in performance of the proposed functions studied in vivo using transgenic worms containing the stem-containing region fused to a reporter gene.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM030870-08
Application #
3278758
Study Section
Genetics Study Section (GEN)
Project Start
1989-09-01
Project End
1992-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
8
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Indiana University Bloomington
Department
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47402
Page, B D; Zhang, W; Steward, K et al. (1997) ELT-1, a GATA-like transcription factor, is required for epidermal cell fates in Caenorhabditis elegans embryos. Genes Dev 11:1651-61
Shim, Y H; Bonner, J J; Blumenthal, T (1995) Activity of a C. elegans GATA transcription factor, ELT-1, expressed in yeast. J Mol Biol 253:665-76
MacMorris, M; Spieth, J; Madej, C et al. (1994) Analysis of the VPE sequences in the Caenorhabditis elegans vit-2 promoter with extrachromosomal tandem array-containing transgenic strains. Mol Cell Biol 14:484-91
Conrad, R; Liou, R F; Blumenthal, T (1993) Conversion of a trans-spliced C. elegans gene into a conventional gene by introduction of a splice donor site. EMBO J 12:1249-55
Spieth, J; Brooke, G; Kuersten, S et al. (1993) Operons in C. elegans: polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 73:521-32
Conrad, R; Liou, R F; Blumenthal, T (1993) Functional analysis of a C. elegans trans-splice acceptor. Nucleic Acids Res 21:913-9
MacMorris, M; Blumenthal, T (1993) In situ analysis of C. elegans vitellogenin fusion gene expression in integrated transgenic strains: effect of promoter mutations on RNA localization. Gene Expr 3:27-36
Broverman, S; MacMorris, M; Blumenthal, T (1993) Alteration of Caenorhabditis elegans gene expression by targeted transformation. Proc Natl Acad Sci U S A 90:4359-63
MacMorris, M; Broverman, S; Greenspoon, S et al. (1992) Regulation of vitellogenin gene expression in transgenic Caenorhabditis elegans: short sequences required for activation of the vit-2 promoter. Mol Cell Biol 12:1652-62
Spieth, J; Shim, Y H; Lea, K et al. (1991) elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family. Mol Cell Biol 11:4651-9

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