Isolation and characterisation of a testis-expressed developmentally regulated gene from the distal inversion of the mouse t-complex

We differentially screened a pool of mouse testis clones in order to identify genes important in germ cell development. One of the isolated clones was found to be expressed only in the male germ line where it is first detected at around the pachytene spermatocyte stage. This gene maps to a subregion of the t-complex in the distal inversion near, but not within, the tw18 and the th20 deletions. A comparison of the t and wild forms of the gene reveals a high degree of sequence conservation. This gene is associated with a CpG-rich island at its 5' end. It encodes a novel protein with extensive alpha-helical structure indicative of coiled-coil interactions.     

Construction of a chromosome 15-specific linking library and identification of potential gene sequences

We have tested a strategy for the construction of a total restriction fragment map of a human chromosome and the rapid isolation of a great number of genes from a specific chromosome. The strategy is based on the cloning of chromosome-specific CpG-rich DNA sequences which are present at the 5' end of many genes. This approach has two important implications: (i) the clones can be used to probe pulsed-field gradient gel blots and link restriction fragments generated by CpG restriction endonucleases, and (ii) the finding of tight genetic or physical linkage between one of these gene probes and a given hereditary disease would make the marker a good candidate gene for this disease. We have constructed a chromosome 15-specific linking library and identified potential gene sequences.     

The nucleotide sequence of a nematode vitellogenin gene.

The nematode, Caenorhabditis elegans, contains a family of six genes that code for vitellogenins. Here we report the complete nucleotide sequence of one of these genes, vit-5. The gene specifies a mRNA of 4869 nucleotides, including untranslated regions of 9 bases at the 5' end and 51 bases at the 3' end. Vit-5 contains four short introns totalling 218 bp. The predicted vitellogenin, yp170A, has a molecular weight of 186,430. At its N terminus it is clearly related to the vitellogenins of vertebrates. However, the vit-5-encoded protein does not contain a serine-rich sequence related to the vertebrate vitellin, phosvitin. In fact, the amino acid composition of the nematode protein is very similar to that of the vertebrate protein without phosvitin. Vit-5 has a highly asymmetric codon choice dictionary. The favored codons are different from those favored in other organisms, but are characteristic of highly expressed C. elegans genes. The strong selection against rare codons is not as great near the 5' end of the gene; rare codons are 15 times more frequent within the first 54 bp than in the next 4.8 kb.     

Characterization of genes encoding rat tonin and a kallikrein-like serine protease

Tissue kallikreins are a group of serine proteases which may function as peptide hormone processing enzymes. Two rat kallikrein genomic clones (RSKG-5 and RSKG-50) were sequenced and characterized. The rat tonin gene and a kallikrein-like gene were found in clones RSKG-5 and RSKG-50, respectively. The tonin gene is 4146 base pairs in length, with both the variant CCAAA and TTTAAA boxes in the 5'-end region and an AATAAA polyadenylation signal at the 3' end of the gene. It has five exons which are separated by four introns. Sequence analysis of 3.7-kb 5' upstream and 7.5-kb 3' downstream of the tonin gene failed to reveal a second kallikrein gene. Sequence comparisons of the RSKG-5 exons with tonin cDNA revealed that only one base in the 3'-noncoding region was different from that in the previously reported rat tonin cDNA. Characteristic TC- and TG-repeated sequences were also found in the first and second introns of the tonin gene. The tonin gene encodes a preprotonin of 259 amino acids (aa). The active enzyme consists of 235 aa and is preceded by a deduced signal peptide of 17 aa and a profragment of 7 aa. Northern blot analysis indicates that RSKG-5 is expressed in a sex-dependent manner in rat submandibular gland, with a higher level expressed in males. The RSKG-50 gene was truncated at an EcoRI site in the second intron, excluding its 5' end. Compared to the coding sequence of pancreatic kallikrein, 12 nucleotides have been deleted in exon 3 of the RSKG-50 gene. The nucleotide sequences of the third, fourth, and fifth exons of the RSKG-50 gene encode a polypeptide of 188 aa residues. The translated peptide is 80% homologous to rat pancreatic kallikrein and 75% homologous to rat tonin in the corresponding regions. Key residues in the RSKG-50 gene product indicate a serine protease with kallikrein-like cleavage specificity at basic amino acids.     

Assignment of the human homologue of the mouse t-complex gene TCTE3 to human chromosome 6q27.

The gene TCTE3 from the mouse t-complex region is expressed specifically in testicular germ cells. It maps in the central subregion of the t-complex on mouse chromosome 17 containing loci involved in transmission ratio distortion and male sterility. In this study, somatic cell hybrid lines have been used to map the human homologue, TCTE3, to the long arm of chromosome 6. CISS hybridization with the human lambda clone h117 refined this chromosome assignment to the very distal position of chromosome 6q27, thus providing further evidence that loci from the t-complex of mouse chromosome 17 can map to opposite arms of human chromosome 6.     

Structure of a cluster of mouse histone genes.

The four mouse histone genes (2 H3 genes, an H2b gene and an H2a gene) present in a cloned 12.9 kilobase fragment of DNA have been completely sequenced including both 5' and 3' flanking regions. These genes are expressed in cultured mouse cells and the 3' and 5' ends of the mRNA have been determined by S1 nuclease mapping. These genes code for a minor fraction of the histone mRNAs expressed in cultured mouse cells. They comprise at most 5-8% of the total histone mRNA of each type. The two H3 genes code for H3.2 and H3.1 histone proteins, while the H2b gene codes for an H2b.1 protein with a single amino acid change (val-leu) at position 18. Only the 3' portion of the H2a gene is contained in the clone and there is an amino acid change (alanine-proline) at position 126. Comparison of the 5' and 3' flanking sequences reveals a conserved sequence at the 3' end of the mRNA which forms a hairpin loop structure. The codon usage in the genes is non-random and there has been no discrimination against CG doublets in the coding region of the genes.     

Control of mouse U1a and U1b snRNA gene expression by differential transcription.

The expression of mouse embryonic U1 snRNA (mU1b) genes is subject to stage- and tissue-specific control, being restricted to early embryos and adult tissues that contain a high proportion of stem cells capable of further differentiation. To determine the mechanism of this control we have sought to distinguish between differential RNA stability and regulation of U1 gene promoter activity in several cell types. We demonstrate here that mU1b RNA can accumulate to high levels in permanently transfected mouse 3T3 and C127 fibroblast cells which normally do not express the endogenous U1b genes, and apparently can do so without significantly interfering with cell growth. Expression of transfected chimeric U1 genes in such cells is much more efficient when their promoters are derived from a constitutively expressed mU1a gene rather than from an mU1b gene. In transgenic mice, introduced U1 transgenes with an mU1b 5' flanking region are subject to normal tissue-specific control, indicating that U1b promoter activity is restricted to tissues that normally express U1b genes. Inactivation of the embryonic genes during normal differentiation is not associated with methylation of upstream CpG-rich sequences; however, in NIH 3T3 fibroblasts, the 5' flanking regions of endogenous mU1b genes are completely methylated, indicating that DNA methylation serves to imprint the inactive state of the mU1b genes in cultured cells. Based on these results, we propose that the developmental control of U1b gene expression is due to differential activity of mU1a and mU1b promoters rather than to differential stability of U1a and U1b RNAs.     

Fusions to the 5' end of a gene encoding a two-domain analogue of staphylococcal protein A.

A novel gene fusion system has been constructed for fusions to the 5' end of gene zz, encoding a two-domain analogue of staphylococcal protein A designated ZZ. Four different genes were fused to the 5' end of zz, and their gene products were analyzed. One of the genes encodes a protein located intracellularly in Escherichia coli and the other three genes encode gene products destined for secretion across the cytoplasmic membrane by the presence of an amino terminal signal sequence. After production in E. coli, the fusion proteins were purified in a single step by IgG-affinity chromatography. The purified ZZ fusions could be used directly for amino terminal sequencing to confirm the start of translation of the intracellular product and the processing of the signal peptide of the translocated products. This is the first example of ZZ fusions to the C-terminus of gene products. To simplify the general use of fusions to the 5' end of zz, a new plasmid vector was constructed containing a multi restriction enzyme cloning linker and the lacZ' gene which enables screening for production in alpha-complementing supE strains of E. coli on indicator plates.