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.     

Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

Tissue-specific expression of rat aldolase A mRNAs. Three molecular species differing only in the 5'-terminal sequences

Three species of aldolase A mRNA (mRNAs I, II, and III) only differing in the structure of the 5'-terminal noncoding region were detected in rat tissues. The cDNA clones for mRNAs II and III were prepared from ascites hepatoma AH60C and sequenced. The mRNA II is 1393 nucleotides long excluding poly(A) tail, while the mRNA III is 1440 nucleotides long, some 50 nucleotides longer than the mRNA II. The mRNAs II and III differ in the sequence between -25 and the 5' termini from the previously reported skeletal muscle aldolase A mRNA (mRNA I, 1343 nucleotides long). By contrast, the residual 5' noncoding sequence (-24 to -1) and the coding and 3' noncoding sequences are common to all the mRNAs. By dot spot hybridization and S1 mapping the distribution of these mRNAs in the various tissues was determined. The mRNA I appears exclusively in a skeletal muscle and some in heart and hepatoma AH60C, whereas the mRNAs II and III appear more or less in all the tissues examined, implying that their appearances are under tissue-specific control. Furthermore, partial nucleotide sequence analysis of the fetal liver aldolase A mRNA supports that aldolase A mRNA that reappeared in hepatoma is really a resurgence of the gene product expressed in the fetus.     

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.     

Molecular characterization of rat substance K receptor and its mRNAs

The nucleotide sequence and the amino acid sequence for rat substance K receptor were deduced by molecular cloning and sequence analysis of its cDNAs. The rat substance K receptor consists of 390 amino acid residues and belongs to the family of G protein-coupled receptors. The comparison of the amino acid sequences of the rat and bovine substance K receptors indicated that they are highly homologous in the regions covering seven putative transmembrane domains, and this similarity is particularly remarkable in the transmembrane segments III and VII and their surrounding regions. RNA blot hybridization analysis showed that the rat substance K receptor is encoded by two species of mRNAs which differ in the lengths of the extreme 5' sequence of the 5'-untranslated regions. This analysis also indicated that the substance K receptor mRNAs are expressed in the gastrointestinal tract. Interestingly, no appreciable substance K receptor mRNAs were detected in poly(A)+ RNAs isolated from the brain and spinal cord, even though these tissues are known to not only contain substance K but also express the mRNA encoding the substance K precursor.     

Rat beta casein cDNA: sequence analysis and evolutionary comparisons.

The complete sequence of a 1072 nucleotide rat beta-casein cDNA insertion in the hybrid plasmid pC beta 23 has been determined. Primer extension was employed to determine the sequence of an additional 82 5'-terminal nucleotides in beta-casein mRNA. Rat beta-casein mRNA consists of a 696 nucleotide coding region, flanked by 52 nucleotide 5' and 406 nucleotide 3' noncoding regions, including a 40 nucleotide poly(A) tail. The derived 216 amino acid sequence of rat beta-casein was compared to the previously determined sequences of beta-caseins from several other species. Approximately 38% of the amino acids have been conserved among the rat, ovine, bovine and human sequences and these conserved amino acids occurred in clusters throughout the protein. One such cluster containing the majority of the potential casein phosphorylation sites was located near the amino terminus. Contrary to the considerable divergence observed for the processed beta-casein, 14 of 15 amino acids in the signal peptide sequence of the precasein were identical between the rat and ovine caseins.     

Fetal and variant alpha-fetoproteins are encoded by mRNAs that differ in sequence at the 5' end

The temperature-sensitive RLA209-15 fetal rat hepatocyte line grown at the nonpermissive temperature (40 degrees C, normal phenotype) produces authentic rat alpha-fetoproteins (AFPs) of 69K and 73K (fetal AFPs) which are encoded by a 2.2-kb mRNA. These cells also produce low levels of a 1.7-kb AFP mRNA and a 65K variant AFP when grown at the permissive temperature (33 degrees C, transformed phenotype). Hybrid-selected translation demonstrates that the 1.7-kb AFP mRNA encodes the 65K variant AFP. Northern blot hybridization and S1 nuclease analyses indicate that the 1.7-kb mRNA lacks sequences present in the first seven 5' exons of the 2.2-kb AFP mRNA. However, the 1.7- and 2.2-kb AFP mRNAs share common sequences extending from the beginning of the eighth exon (corresponding to nucleotide 873 of the fetal AFP mRNA) to the 3' end. Primer extension analysis suggests that the 1.7-kb RNA contains additional sequences 5' to the common regions shared by both AFP mRNAs. We have previously shown that adult rat liver produces a 1.7-kb AFP mRNA; we now report the isolation of a cDNA (ARFP5) encoding this variant AFP mRNA from an adult rat liver cDNA library. Restriction endonuclease mapping and sequence analysis of ARFP5 confirm that the 1.7- and 2.2-kb AFP mRNAs share similar sequences at the 3' region (approximately 1.1 kb). However, ARFP5 contains an additional 90 bp variant AFP mRNA-specific 5' sequence which is located in the seventh intron of the rat AFP gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence of rat intestinal vitamin D-dependent calcium-binding protein derived from a cDNA clone. Evolutionary implications

We have recently reported molecular cloning of the cDNA synthesized from rat duodenal mRNA-encoding intestinal calcium-binding protein (ICaBP), a vitamin D3-induced protein (Desplan, C., Thomasset, M., and Moukhtar, M. S. (1983) J. Biol. Chem. 258, 2762-2765). Nucleotide sequence analysis of the longest cDNA insert (375 base pairs) permitted the assignment of 207 nucleotides of the coding region and 104 nucleotides of the entire 3'-noncoding region of the mRNA. Although the derived amino acid sequence for rat ICaBP differed from the bovine and porcine sequences by 16 and 14 residues, respectively, all the residues of each calcium-binding site met the proposed requirements of the "EF hand" theory. In contrast, several differences found in the linker regions might explain the absence of cross-immunoreactivity between rat and porcine ICaBPs. Analysis of nucleotide sequence homologies between the coding and noncoding regions showed that the region coding for the two calcium-binding sites (I and II) was immediately followed in the noncoding region by a sequence very similar to the sequence coding for site I. This suggests that rat ICaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure and that low Mr ICaBP could result in early termination of the translation of a larger molecule containing four sites.     

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.     

Expression of the genes coding for the skeletal muscle and cardiac actions in the heart

Several types of evidence indicate that the gene coding for the skeletal muscle actin is expressed in the rat heart: 1) A recombinant plasmid containing an insert with a nucleotide sequence identical to that of the homologous region of skeletal muscle actin gene was isolated from a cDNA library prepared on rat cardiac mRNA template. 2) Using specific probes it was found that the hearts of newborn rats contain a significant amount of skeletal muscle actin mRNA. The quantity of this mRNA in the heart decreases during development. 3) The skeletal muscle actin gene is DNAase I sensitive in nuclei from rat heart tissue. A plasmid containing a cDNA insert homologous to a part of the cardiac actin mRNA was isolated and sequenced. It was found that in spite of the great similarity between the amino acid sequence of the skeletal muscle and cardiac actins, the nucleotide sequences of the two mRNAs are considerably divergent. There is only limited sequence homology between the 3' untranslated regions of the two mRNAs. However, there is an extensive sequence homology between the 3' untranslated regions of the rat and human cardiac mRNAs, suggesting a functional role for this region of the gene or mRNA.     

Expression of three mRNA species from a single rat aldolase A gene, differing in their 5' non-coding regions

The complete nucleotide sequence of the rat aldolase A isozyme gene, including the 5' and 3' flanking sequences, was determined. The gene comprises ten exons, spans 4827 base-pairs and occurs in a single copy per haploid rat genome. The genomic DNA sequence was compared with those of three species of rat aldolase A mRNA (mRNAs I, II and III) that have been found to differ from each other only in the 5' non-coding region and to be expressed tissue-specifically. It revealed that the first exon (exon M1) encodes the 5' non-coding sequence of mRNA I, while the second exon (exon AH1) encodes those of mRNAs II and III and the following eight exons (exons 2 to 9) are shared commonly by all the mRNA species. These results allowed us to conclude that mRNA I and mRNAs II, III were generated from a single aldolase A gene by alternative usage of exon M1 or exon AH1 in addition to exons 2 to 9. S1 nuclease mapping of the 5' ends of their precursor RNAs suggested that these three mRNA species were transcribed from three different initiation sites on the single gene.     

UTRdb and UTRsite: specialized databases of sequences and functional elements of 5' and 3' untranslated regions of eukaryotic mRNAs

The 5' and 3' untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb, a specialized database of 5' and 3' untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements. All internet resources implemented for retrieval and functional analysis of 5' and 3' untranslated regions of eukaryotic mRNAs are accessible at http://bigarea.area.ba.cnr.it:8000/EmbIT/UTRH ome/