Characterization of a gene encoding a basic protein of the spermatid nucleus, TNP2, and its close linkage to the protamine genes in the bull.

During elongation and condensation of the spermatid nucleus, histones are replaced by spermatid-specific transition proteins (TNP). TNP1 is well characterized at the cDNA and at the genomic level and was found to be highly conserved during mammalian evolution (similarity between 83 to 98%). We here describe for the first time the nucleotide sequence and organization of the gene for TNP2. The gene was isolated from a bull cosmid library and was found to contain a single intron of 910 bp. The coding sequence consists of 390 bp and has a similarity of about 70% to that of the TNP2 cDNAs of mouse and rat. At the basis of amino-acid sequences, the bull TNP2 is 14 and 15 amino acids longer than that of mouse and rat, respectively, and the similarity is only 45% between bull and mouse and 42% between bull and rat. However, the evolutionary divergence has not occurred at the cost of basic amino acids which are of functional importance in DNA-protein interaction in the condensing spermatid nucleus. The TNP2 gene is closely linked to the protamine genes in the bull genome.     

The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis

A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.     

Molecular cloning and chromosomal mapping of the human gene for the testis-specific catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A).

A cDNA for an alternatively spliced variant of the testis-specific catalytic subunit of calmodulin dependent protein phosphatase (CaM-PrP) was cloned from a human testis library. The nucleotide sequence of 2134 base pairs (bp) encodes a protein of 502 amino acids (Mr approximately 57,132) and pI 7.0. The cDNA sequence differs from the murine form of this gene by a 30 bp deletion in the coding region, the position of which matches those in the two other genes for the catalytic subunit. These data indicate that this alternative splicing event arose prior to the divergence of the three genes. The deduced sequence of the human protein is only 88% identical to the homologous murine form, in striking contrast to the other two CaM-PrP catalytic subunits which are highly conserved between mouse and human (approximately 99%); this indicates a more rapid rate of evolution for the testis-specific gene. Analysis of Southern blots containing DNA from human-hamster somatic cell hybrids show that the gene is on human chromosome 8.     

人甲状腺受体相互作用蛋白15基因全长cDNA的克隆及其特性

通过生物信息学分析及RT PCR技术 ,从人垂体cDNA文库中克隆到甲状腺素受体相互作用蛋白 15(hTRIP15)的全长cDNA ,长度 1963bp ,编码 4 4 3氨基酸 ,同时克隆该基因不同剪接方式所形成的新的异构体 ,长度 1984bp ,编码 4 50氨基酸 .与基因组序列比较显示该基因具有 12个外显子 ,5号外显子 3′端具有 2个剪切的接点 (-ag) .搜寻UniGene数据库作染色体定位于D15S146 D15S117,该基因在生物进化上具有较高的保守性 ,从单细胞藻类到人类均有该基因同源物表达 ,亚细胞定位为核内 .Northern杂交显示 ,该基因具有 3种不同大小的转录本 ,分别约为 2 0、3 5及 4 0kb ,且在人体各组织中均有一定表达 ,其中骨骼肌、心脏及肾脏组织为高表达 .半定量RT PCR显示在一些内分泌组织均有表达 ,以肾上腺较高 .     

Cloning and Sequence Analysis of a cDNA from Seminal Vesicle Tissue Encoding the Precursor of the Major Protein of Bull Semen

Abstract

A cDNA library derived from poly(A)⁺RNA of bull seminal vesicle- tissue was screened with a synthetic DNA hybridisation probe specific for the major protein of bull semen. A positive clone pMP17, containing a 680 bp insert, was sequenced. In combination with primer extension sequencing of poly(A)⁺RNA, a DNA sequence of 700 bp was determined. This DNA encoded a reading frame for 134 amino acids, starting with an ATG and terminated by a TAG codon. This comprised 25 amino acids of a signal peptide followed by 109 amino acids with the known sequence of the major protein.     

新型小鼠淋巴组织特异性肌动蛋白结合蛋白相关序列cDNA克隆

 应用抑制性差减杂交技术 ( SSH)克隆两种不同小鼠胸腺基质细胞的差异表达基因 ,获得新基因片段 C55.通过 Gen Bank检索及 RT- PCR扩增出一个全长 1 .4kb的 c DNA.杂交分析认为它是一个完整的 c DNA序列 .c DNA序列分析表明 ,它拥有一个 636bp的开放读码框架 ,编码 2 1 2个氨基酸 .同源序列比较发现 ,它编码一个肌动蛋白相关蛋白的新成员 ,该序列与多种已知的肌动蛋白相关蛋白 SM2 2 α及其同源蛋白在氨基酸水平上有 62 %～ 95%的同源性 .Northern杂交分析显示 ,该基因 m RNA转录本在两种不同胸腺基质细胞中的表达存在显著差异 . RT- PCR分析显示 ,该基因特异表达于小鼠淋巴相关组织中 ,而在非淋巴组织中无表达 .     

Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse

We have recently cloned a cDNA encoding mitochondrial porin in Drosophila melanogaster and shown its chromosomal localization (Messina et al., FEBS Lett. (1996) 384, 9–13). Such cDNA was used as a probe for screening a genomic library. We thus cloned and sequenced a 4494-bp genomic region which contained the whole gene for the mitochondrial porin or VDAC. It was found that this D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3′ untranslated sequence (3′-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5′-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5′-UTR sequence, demonstrated to be transcribed by 5′-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2–1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites.     

Molecular cloning of chicken metallothionein. Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA.

A cDNA library was constructed using RNA isolated from the livers of chickens which had been treated with zinc. This library was screened with a RNA probe complementary to mouse metallothionein-I (MT), and eight chicken MT cDNA clones were obtained. All of the cDNA clones contained nucleotide sequences homologous to regions of the longest (376 bp) cDNA clone. The latter contained an open reading frame of 189 bp, and the deduced amino acid sequence indicates a protein of 63 amino acids of which 20 are cysteine residues. Amino acid composition and partial amino acid sequence analyses of purified chicken MT protein agreed with the amino acid composition and sequence deduced from the cloned cDNA. Amino acid sequence comparisons establish that chicken MT shares extensive homology with mammalian MTs, but is more closely related to the MT-II than to the MT-I isoforms from various mammals. The nucleotide sequence of the coding region of chicken MT shares approximately 70% homology with the consensus sequence for the mammalian MTs. Southern blot analysis of chicken DNA indicates that the chicken MT gene is not a part of a large family of related sequences, but rather is likely to be a unique gene sequence. In the chicken liver, levels of chicken MT mRNA were rapidly induced by metals (Cd2+, Zn2+, Cu2+), glucocorticoids and lipopolysaccharide. MT mRNA was present in low levels in embryonic liver and increased to high levels during the first week after hatching before decreasing again to the basal levels found in adult liver. The results of this study establish that MT is highly conserved between birds and mammals and is regulated in the chicken by agents which also regulate expression of mammalian MT genes. However, in contrast to the mammals, the results suggest the existence of a single isoform of MT in the chicken.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1-450 and P3-450

Polysome immuno-adsorption, with immunoglobulin G directed against two 3-methylcholanthrene-induced mouse liver cytochrome P-450 proteins, was used to enrich mRNA from 3-methylcholanthrene-treated C57BL/6N mouse liver. cDNA transcribed from the P-450-enriched mRNA was then cloned into the Okayama-Berg vector. Two cDNA classes were detected upon differential screening of the clone bank with [32P]cDNA derived from 3-methylcholanthrene-induced immuno-enriched versus control mRNA. Several representatives of these two classes were judged to be near full length by comparison with their corresponding mRNA mobilities on denaturing agarose gels. A continuous reading-frame near the 5' end of one cDNA class (P1-450) corresponds to a protein having 15 of 17 residues the same as the published N-terminal sequence of rat P-450c. A continuous reading frame near the 5' end of the other class (P3-450) corresponds exactly to the first 25 amino acids of the published N-terminal sequence of rat P-450d. The P1-450 cDNA is at least 700 bp longer than the P3-450 cDNA. Heteroduplex analysis and Southern blot hybridization demonstrate that these mRNAs share approx. 1100 bp of sequence homology. Genomic P1-450 and P3-450 clones were isolated from a gene library constructed from C57BL/6N mouse liver DNA. By heteroduplex analysis with the corresponding cDNA, the P1-450 gene spans about 6 kb and the P3-450 gene about 7 kb. The intron-exon patterns are very similar, with the second and seventh exons being much larger than the other five. The 3' terminal exon of P1-450 is about 500 bp longer than that of P3-450. These data suggest that both P1-450 and P3-450 have diverged from a common ancestral gene.     

Cloning of cDNA for a novel fibrinogen/angiopoietin-related protein, FARP

Using a low abundant gene screening strategy in the human dermal papilla cell cDNA library, we isolated a novel cDNA, which was 1,872 bp of nucleotides in length and contained an open reading frame encoding 405 amino acids. We designated it 'fibrinogen/angiopoietin-related protein' (FARP) as it contained the characteristic coiled-coil domain and fibrinogen-like domain in the NH2- and COOH-terminal, which are conserved in angiopoietins. FARP has a highly hydrophobic region at the N-terminus that is typical of a secretory signal sequence. Recently, a very similar gene, HFARP, was cloned and they have a difference of only 18 amino acids in N-terminus. While HFARP was expressed only in the liver, northern blot analysis showed that FARP mRNA is abundantly expressed in the liver, placenta, prostate, and ovary in human adult tissues. It was also expressed in the fetal liver and lung carcinoma cell line. Further study will be needed to clarify the function of the FARP gene.