Genomic organization of the human gamma adducin gene

We report the genomic structure of the human gamma adducin gene (ADD3). Adducin is a protein involved in cytoskeletal assembly and composed of alpha-beta or alpha-gamma subunits which share a high degree of homology between human and rat. Mutations in alpha subunit have been shown associated to both human and rat hypertension. The human ADD3 gene spans over 20 kb and is composed of at least 13 introns and 14 exons covering the entire coding region. The exon size ranges from 81 bp to greater than 293 bp and the intron size from 111 bp to longer than 3.2 kb. We also demonstrate the presence of an alternative splicing event around exon 13, whose sequence, position, and expression is analogous in rat Add3 gene. Moreover, human ADD3 amino acid sequence presents 91.9% of identity compared to rat sequence. Characterization of human ADD3 gene provides an important tool for mutation analysis.     

cDNA sequence coding for a rat glia-derived nexin and its homology to members of the serpin superfamily

Rat glial cells release a neurite-promoting factor with serine protease inhibitory activity. By using a rat glioma cDNA clone as a probe, it was possible to isolate rat cDNAs containing the entire sequence coding for this neurite-promoting factor. The largest rat cDNA (approximately 2100 bp) was characterized by DNA sequencing. It contained the entire coding region, 135 bp of the 5' nontranslated region, and about 750 bp of the 3' nontranslated region. The open reading frame coded for 397 amino acids including a putative signal peptide of 19 amino acids. The correct identity of the coding sequence was substantiated by the fact that the sequence of tryptic peptides, derived from the purified rat factor, matched exactly with the deduced amino acid sequence. The rat protein sequence had 84% homology with the corresponding protein from human glioma cells. Both amino acid sequences indicated that the proteins belong to the protease nexins [Baker, B.J., Low, D. A., Simmer, R. L., & Cunningham, D.D. (1980) Cell (Cambridge, Mass.) 21, 37-45] and therefore can be defined as glia-derived nexins (GDNs). Further analysis showed that both rat and human GDN belong to the serpin superfamily and share 41%, 32%, and 25% homology with human endothelial-cell-type plasminogen activator inhibitor, antithrombin III, and alpha-1 proteinase inhibitor, respectively.     

Filaggrin, an intermediate filament-associated protein: structural and functional implications from the sequence of a cDNA from rat.

Filaggrin is an intermediate filament-associated protein that is involved in aggregation of keratin filaments in fully cornified cells of the mammalian epidermis, and is an important marker for epidermal differentiation. In this report, the sequence of a rat cDNA clone coding for a portion of the polymeric precursor, profilaggrin, is presented. The cDNA is 2,314 bp long with 1,875 bp of coding region ending with an A-T-rich 3' noncoding region. Genomic analysis indicates that the profilaggrin gene consists of 20 +/- 2 repeats of 1,218 bp of sequence coding for 406 amino acids, making the mRNA at least 25-27 kb in length. Each repeat consists of a filaggrin domain and a linker sequence with an estimated size of 380 and 26 amino acids, respectively. High levels of profilaggrin mRNA are found only in keratinizing epithelia. Comparison of the rat filaggrin sequence with that of mouse and human filaggrin and with the sequence of phosphorylated peptides from mouse profilaggrin indicates that the proteins share extensive amino acid sequence similarities, especially in the two phosphorylated regions. Proteolytic processing sites are also quite similar in rat and mouse. The three species show blocks of sequence that are similar in length and composition which alternate with sequences that are variable in length. This analysis suggests that the evolution of the present-day filaggrins has been constrained by maintenance of phosphorylation sites and overall amino acid composition. The cDNAs for the profilaggrins are similar in structure, reflecting genes that have simple repeating structures and lack introns within their coding regions. Mouse and rat profilaggrin terminate with a nonpolar sequence atypical of the rest of the coding region, and have similar 3' noncoding regions. To explain these observations, a novel evolutionary model is proposed.     

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.     

大鼠脑红蛋白基因编码区的克隆、多态性分析及该基因组织表达谱研究

参考人和小鼠脑红蛋白（Neuroglobin,NGB)的cDNA序列设计简并引物,用RT－PCR方法从大鼠脑组织中扩增出大鼠NGB基因编码区的cDNA序列,该序列与小鼠NGB基因编码区的序列同尖性为96％,与人NGB基因编码区的序列同源性为88％,进一步分析表明,大鼠NGB基因编码区存在多个多态性位点;113t/c[138P],133a/g[N45D],388a/g[R130G],417t/c.该序列已被GenBank接受,登录号为AF333245,RT－PCR分析表明,该基因在大鼠脑,肝,肾,心肌和骨骼肌中均有较高水平表达,提示了其功能上的重要性。     

Cloning, expression and sequence homologies of cDNA for human gamma enolase

The nucleotide sequence of the human gamma-enolase mRNA was determined from recombinant cDNA clones. The sequence spans 2273 bp and includes the complete coding region of 1299 bp, a 5'-noncoding region of 74 bp and a 897-bp-long 3'-noncoding region containing a variant polyadenylation signal (ATTAAA). The deduced amino acid (aa) sequence is 433 aa long and shows a 97% similarity with rat gamma-enolase. Both the 5'- and 3'-untranslated regions are similar (82% and 68%, respectively) to the analogous regions of the rat gamma-enolase gene, suggesting that a strong selective pressure operates on noncoding segments of gamma-enolase mRNAs. The size of the gamma-enolase mRNA expressed in human brain is 2.4 kb. A crosshybridizing 1.5-kb message is detected in human skeletal muscle which may be derived from the beta-enolase-coding gene.     

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.     

Isolation and characterization of the human ornithine transcarbamylase gene: structure of the 5'-end region

We isolated over 20 phage clones carrying the ornithine transcarbamylase (OTC) [EC 2.1.3.3] gene, from two independently constructed human genomic DNA libraries, using as probes either a rat OTC cDNA or several nuclear DNA fragments derived from some of these isolated clones. These clones, classified into 10 different groups, overlapped and spanned a region of more than 85 kilobase pairs of the human genomic DNA. Restriction mapping and Southern blot analyses demonstrated that one of the clones covers the 5'-end region of the OTC gene. We sequenced the 5'-end region of the OTC gene and found that it covered 665 base pairs of the 5'-flanking region, the complete first exon and a part of the first intron (150 base pairs). In the 5'-flanking region, there were two pairs of putative CAAT and TATA boxes and one enhancer core-like sequence, GTGGAAAG. The first exon contained a coding region for most of the OTC presequence, i.e. 26 out of 32 amino acid residues of the presequence, including the initiation methionine.     

Cloning and characterization of the human and rat islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) genes

Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and rat IGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRP gene has five exons of similar length and coding sequence to the mouse IGRP gene and is located on human chromosome 2q28-32 adjacent to the myosin heavy chain 1B gene. In contrast, the rat IGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissue-restricted expression of the IGRP gene and the identification of key amino acid sequences that determine its biological activity.     

Complete nucleotide sequence of human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) cDNA and a comparison with human and rat PRPS gene families.

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) were isolated from a glioblastoma cell line MGC 1 cDNA library. The longest clone contained 2,075 base pairs (bp) almost covering the 2.3-kb mRNA and the base sequence of the coding region (954 bp) had a 92.0% sequence homology with that of rat PRS I cDNA. The deduced amino acid sequences were identical between human and rat PRS I. This perfect conservation has heretofore not been reported for other enzymes involved in nucleotide metabolism and glycolysis. A comparison with other isoforms of this enzyme, PRS II and PRS III, showed that the human PRS I was 79.9 and 92.2% homologous in the coding sequence and 95.3 and 94.0% in the deduced amino acid sequence to human PRS II and PRS III, respectively. The high value of the synonymous difference between PRS I and PRS II cDNAs places their time of divergence long before that of the radiation of mammals. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago.