Nonallelic members of the cytochrome c multigene family of the rat may arise through different messenger RNAs

We determined the nucleotide sequences of three nonallelic cytochrome c genes (from recombinant clones Ch4A-RC5, 6 and 8) isolated from the rat cytochrome c gene family. In contrast with a fourth gene (from Ch4A-RC4), which has an intron and correctly encodes rat cytochrome c, these three appear to be pseudogenes and resemble mRNA molecules in two respects: they are all missing the intron of clone 4, and sequence homology with clone 4 in their 3' noncoding regions abruptly ends at two different A-rich tracts reminiscent of poly(A) tails. We also detect three cytochrome c mRNAs of sizes 1400, 1100 and 700 nucleotides in several tissues of the adult rat. The size differences among the mRNAs can be accounted for by length heterogeneity in their 3' noncoding regions. Two of the 3' ends map to the two points where the mRNA-like genes diverge from clone 4 at poly(A) tracts. Furthermore, short direct repeats flank the genes of clones 5, 6 and 8 at the positions where their sequences diverge. The observations suggest that these members of the cytochrome c multigene family may arise through insertion into the genome of DNA copies of cytochrome c mRNAs.     

Molecular cloning of a human co-beta-glucosidase cDNA: evidence that four sphingolipid hydrolase activator proteins are encoded by single genes in humans and rats

Authentic cDNAs encoding the activator protein for acid beta-glucosidase (EC3.2.1.45), co-beta-glucosidase, were cloned from the pCD and lambda gt11 human cDNA libraries. Initial screening with oligonucleotide mixtures encoding amino acid sequences of co-beta-glucosidase identified partial cDNAs which were used to obtain a potentially full-length cDNA from the lambda gt11 library. This clone (2767 bp), EGTISI, contained 5' (38 bp) and 3' (1157 bp) noncoding sequences, a translation initiation site, and an open reading frame encoding 524 amino acids which included a typical hydrophobic signal sequence (16 amino acids). Computer analyses identified three regions of high similarity to co-beta-glucosidase encoded by tandem sequences in EGTISI. Searches revealed that two of these regions encoded peptides of known function; SAP1 (sphingolipid activator protein 1) and protein C (a new sphingolipid activator protein) were encoded by EGTISI sequences 5' and 3', respectively, to those for co-beta-glucosidase. The third region of similarity, encoding a theoretical peptide (undefined function), was located most 5' in the cDNA. EGTISI and its encoded polypeptide had high similarity (77% nucleotide identity and about 80% amino acid similarity) to a rat Sertoli cell cDNA and its encoded sulfated glycoprotein-1. These results indicate that a single highly conserved gene encodes the precursor for four potential sphingolipid activator proteins in rat and man.     

Comparison of the whey acidic protein genes of the rat and mouse.

Whey acidic protein (WAP), a hormonally-regulated 14,000 dalton cysteine-rich protein, is the principal whey protein found in rodent milk. Genomic clones encompassing both the 2.8 Kb rat and 3.3 Kb mouse WAP genes have been characterized. The genes consist of four exons and three introns. The middle two exons encode the two cysteine-rich regions which probably form separate protein domains. Homology in the 5' flanking DNA of the mouse and rat extends at least 325 bp upstream of the putative CAP site, including a precisely conserved stretch of 50 bp around the unusual TATA and CAAT sites. The homology previously observed between the 3' noncoding sequences of the rat and mouse mRNAs extends at least 20 bp into the 3' flanking region. Several potential glucocorticoid receptor binding sites have been found in the 5' flanking region of the WAP gene. The conservation of the 5' flanking region of the WAP genes may be related to regulation of expression of WAP by peptide and/or steroid hormones.     

Complete mitochondrial genome of the longtooth grouper Epinephelus bruneus (Perciformes, Serranidae)

We determined the complete nucleotide sequence of the mitochondrial (mt) genome for the longtooth grouper, Epinephelus bruneus (Perciformes, Serranidae). This mt genome, consisting of 16,686 base pairs (bp), encoded genes for 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region as those found in other vertebrates, with the gene order identical to that of typical vertebrates. A major noncoding region between the trnP and trnF genes (991 bp) was considered to be the control region (D-loop). Within this sequence, 22 copies of a 17-bp tandem repeat element, 5'-TGATATTACATATATGC-3', were identified in the control region unlike previous reported Epinephelus species.     

Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease

The cDNA of mouse pancreatic mRNA has been cloned. After the library was screened with a rat ribonuclease cDNA probe, the positive clones were isolated and sequenced. There were no differences from the previously determined protein sequence. The mRNA codes for a preribonuclease of 149 amino acid residues including a signal peptide of 25 amino acids. The 3' noncoding region has a length of 260 bp, and the total mRNA length is approximately 940 bp. Comparison with the rat pancreatic ribonuclease sequence showed a high rate of nucleotide substitution. Within the coding region, nonsynonymous and synonymous substitution rates are 4.3 X 10(-9) and 15 X 10(-9) nucleotide substitutions/site/year, respectively. The latter value is one of the highest rates observed in the molecular evolution of mammalian nuclear genes. In the signal sequences the synonymous substitution rate is much lower and about the same as the nonsynonymous rate. Signal sequences of other mouse and rat proteins also exhibit little difference between synonymous and nonsynonymous rates. The sequences of rat and mouse pancreatic ribonuclease messengers were compared with those of bovine pancreatic, seminal, and brain ribonuclease. While the 3' noncoding regions of rat and mouse are very similar, as are those of the three bovine messengers, there is no significant similarity between both rodent and the three bovine messengers for the greater part of these regions. There is a duplication of approximately 50 nucleotides in the 3' noncoding region of the bovine messengers, with a region rich in A and C in between. The presence of this structural feature may be correlated with recent gene duplications that have occurred in the bovine genome.     

Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA.     

The rat casein multigene family. Fine structure and evolution of the beta-casein gene

Eight overlapping phage clones, spanning 34.4 kilobase pairs of genomic DNA, containing the 7.2-kilobase pair rat beta-casein gene have been isolated and characterized. The first 510 base pairs (bp) of 5' flanking, 110 bp of 3' flanking, and all the exon/intron junctions have been sequenced. The beta-casein gene contains 9 exons ranging in size from 21 to 525 bp. We have attempted to identify potential regulatory elements by searching for regions of sequence homology shared between milk protein genes which respond similarly to lactogenic hormones and by searching for previously reported hormone receptor-binding sites. Within the conserved first 200 bp of 5' flanking sequences 3 regions of greater than 70% homology were observed between the rat beta- and gamma-casein genes. One of these contains a region 90% homologous to the chicken progesterone receptor-binding site. The conserved 5' noncoding region, the highly conserved signal peptide, and the hydrophobic carboxyl-terminal region of the protein are each encoded by a separate exon. In contrast the evolutionarily conserved phosphorylation site of beta-casein is formed by an RNA-splicing event. The exons which encode the phosphorylation sites of beta-casein appear to have resulted from an intragenic duplication. Based upon the exon structure of the casein genes, an evolutionary model of intragenic and intergenic exon duplications for this gene family is proposed.     

The primary structure of a glyceraldehyde-3-phosphate dehydrogenase gene from Saccharomyces cerevisiae.

The complete nucleotide sequence of the coding, as well as the flanking noncoding regions, of a yeast glyceraldehyde-3-phosphate dehydrogenase gene was determined. Both the 5' and 3' noncoding sequences are extremely AT-rich and regions of partial dyad symmetry are present immediately adjacent to the 5' and 3' ends of the translated portion of the gene. The sequence AAUAAA is present in the 3' noncoding region of this gene and is a part of an extensive region of dyad symmetry which is structurally related to the 3'-terminal portion of both procaryotic mRNAs, as well as some eukaryotic mRNAs. The coding region of this gene does not contain intervening sequences. Establishment of the primary structure of this glyceraldehyde-3-phosphate dehydrogenase gene provides a basis for further studies involving in vitro mutation of the gene and subsequent analysis of gene expression in vivo.     

Cloning of two different 5' untranslated exons of bovine acidic fibroblast growth factor by the single strand ligation to single-stranded cDNA methodology.

In an attempt to characterize the 5' UTR of the aFGF mRNAs we used the new anchored PCR methodology, single strand ligation to ss-cDNAs (SLIC). In bovine brain and retina, two kinds of aFGF cDNA clones were isolated. They contained two alternative exons located 34 bp upstream to the translation initiation codon ATG. Taking into account the number of clones specific for each exon, the two mRNAs are expressed with the same ratio in both tissues. One of these bovine 5' UTR exons (136 bp) showed 81% identity to a human 5' UTR exon, the second one (323 bp) was 70% identical to the second human 5' UTR exon with a central region of 90 nucleotides showing 41% identity. The conservation of the splicing positions for these 5' UTR alternate exons in both bovine and human species, suggests that the overall structure of the aFGF gene is conserved in mammals. Furthermore, the conservation of the nucleotide sequences and of the localization of these 5' UTR exons suggests that these non-coding regions may be involved in the control of aFGF gene expression.     

Complete sequence of ovine alpha s2-casein messenger RNA

The primary structure of mRNA coding for ovine alpha s2 casein has been determined by chemical sequencing of three cDNA clones and the primer extension products of the longest one. The mRNA was 1,024 nucleotides long, excluding the poly(A) tail. The length of the 5' noncoding, coding and 3' noncoding regions was 53, 669 and 302 nucleotides, respectively. A comparison of the nucleotide sequence of ovine alpha s2-casein and guinea-pig casein A mRNAs revealed an extensive homology in the 5' and 3' noncoding regions. The deduced amino acid sequence of ovine alpha s2-casein was compared with its bovine and guinea-pig counterparts. Moreover, an heterogeneity was evidenced in the mRNA population of the alpha s2-casein.