Molecular Cloning of cDNA to mRNA for a Cerebellar Spot 35 Protein

The nucleotide sequence of mRNA for rat cerebellar spot 35 protein, a Ca-binding protein, was determined from recombinant complementary DNA (cDNA) clones. The sequence was composed of 1,714 base pairs (bp) which included the 783 bp of the complete coding region, the 130 bp of the 5'-noncoding region, and the 801 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, a polyadenylic acid [poly(A)] tail was also found. Because the size of spot 35 mRNA was estimated to be about 1,900 bases by Northern blot analysis, the longest insert was verified to contain a nearly full-length cDNA sequence including the poly(A) tail. The amino acid sequence of the protein deduced from the nucleotide sequence contains 261 amino acids and at least five Ca-binding domains. There was a high homology in the amino acid sequences (79%) and the nucleotide sequences (77%) between spot 35 protein and chick intestinal Ca-binding protein (28K).     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

Cloning and nucleotide sequence of the murine hsp84 cDNA and chromosome assignment of related sequences

The nucleotide (nt) sequence of mouse 84-kDa heat shock protein (Hsp) cDNA has been determined using a combination of molecular cloning and oligodeoxynucleotide priming on poly(A) + RNA. The cDNA was 2.5 kb long, not including the poly(A) tail. It contained a 5' leader of about 94 nt that was G + C-rich, and a 243-nt 3'-untranslated region that was A + T-rich in the vicinity of the polyadenylation signal. Gene hsp84 codes for an acidic polypeptide of 724 amino acid (aa) residues. Mouse Hsp84 had 81% and 63% aa homology to Drosophila melanogaster Hsp82 and yeast Hsp90, respectively. The nucleotide sequence had 74% and 59% homology to Drosophila and yeast hsp sequences, respectively, in the coding regions of these genes. This homology did not extend to the 5' - and 3'-untranslated regions. Chromosomal analysis indicated that hsp84-related sequences are on at least three different chromosomes.     

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.     

Vitamin-D dependent 9 kDa calcium-binding protein gene: cDNA cloning, mRNA distribution and regulation

Cholecalciferol (calcitriol) the active hormonal form of vitamin D induces the synthesis of at least two intracellular calcium-binding proteins (Ka = 10(6) M-1), the cholecalcins (CaBP) in mammals. We used the synthesis of these proteins to study the genomic steroid-like action of vitamin D. The 9 kDa CaBP is mainly concentrated in the duodenum while 28 kDa CaBP is located in the kidney and cerebellum. Complementary DNA copies of rat intestinal 9 kDa CaBP mRNA were cloned in E. coli. The deduced amino acid sequence for 9 kDa CaBP contains two 'EF hand' domains corresponding to calcium-binding sites I and II. The homology observed suggests, after comparison with the structures of other intracellular CaBPs, that rat 9 kDa CaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure seen in 28 kDa CaBP from kidney and cerebellum of rat. Northern blots showed that the cDNA sequence hybridizes to a homogeneous 500-600 nucleotide mRNA species from rat duodenum. Larger mRNA species encoding 28 kDa CaBP were undetectable in rat kidney and cerebellum even under low stringency conditions. These findings demonstrate that there is no cross-hybridization between 9 kDa and 28 kDa CaBP mRNAs, and Southern analysis indicates that there are distinct genes coding for each rat cholecalcin. The cDNA probe was used to analyze the specific 9 kDa CaBP gene expression along the intestine of growing rats and during gestation and fetal development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue and species distribution of mRNA encoding two ADP-ribosylation factors, 20-kDa guanine nucleotide binding proteins

Cholera toxin catalyzed ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide binding protein of the adenylyl cyclase system, is enhanced by approximately 20-kDa guanine nucleotide binding proteins, termed ADP-ribosylation factors or ARFs. ARF is an allosteric activator of the A1 catalytic protein of the toxin. Bovine ARF cDNA clones, ARF-1 isolated from adrenal (Sewell & Kahn, 1988) and ARF-2B from retina (Price et al., 1988), exhibit nucleotide and deduced amino acid sequences that are 80% and 96% identical, respectively, in the coding region. To determine tissue and species distribution of ARF-like mRNAs, bovine ARF-2B and human ARF-1 cDNAs and 30- or 48-base oligonucleotide probes that distinguish between ARF-1 and ARF-2B cDNAs in coding and 3'-untranslated regions were used for Northern analysis of poly(A+) RNA from different tissues and species. On the basis of hybridization with specific oligonucleotide probes, all bovine tissues contained mRNAs of 1.7 and 2.1 kb that were related to ARF-1 and ARF-2B, respectively. Northern analysis of brain poly(A+) RNA from different species with ARF-2B and ARF-1 cDNAs at low stringency demonstrated several bands varying in size from 0.9 to 3.7 kb. A 1.7-kb band consistently hybridized with an ARF-1 30-base coding-region probe but not with a probe for the 3'-untranslated region. Similar ARF-2B oligonucleotide probes did not hybridize with rat, mouse, rabbit, or human brain mRNA. Cleavage of ARF-2B cDNA with PvuII generated two fragments, one containing coding and the other 3'-noncoding region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma

Two overlapping clones, covering the entire coding sequence of human M2-type pyruvate kinase (PK) cDNA, were isolated and sequenced. Nucleotide sequencing results showed that they contained the 109-bp 5'-untranslated region, the 1593-bp coding region and the 585-bp 3'-untranslated region. Nucleotide sequence homology was 90% and 69% with rat M2-type and L-type PK cDNA, respectively. In situ hybridization using the human M2-type PK cDNA probe disclosed that the gene for M2-type PK is located at band q22 on chromosome 15. Northern blot analysis with RNA from human hepatoma demonstrated that M2-type PK was predominantly expressed in hepatoma cells, whereas L-type PK was preferentially expressed in the non-tumor portion of the liver.     

Vimentin cDNA clones covering the complete intermediate-filament protein are found in an EHS tumor cDNA library

Intermediate filaments are part of the cytoskeleton of most cells. To analyze changes in intermediate filament synthesis, we have isolated two cDNA clones (pV-C25, pV-C877) that cover the complete coding sequence of the murine intermediate filament protein vimentin. The cDNA clones were isolated from a murine Engelbreth-Holm-Swan (EHS) tumor cDNA library by screening under (i) non-stringent conditions with a synthetic oligodeoxynucleotide (oligo), LW-36, which is specific for type-IV collagen, and (ii) stringent conditions with oligo LW75, which was derived from the vimentin clone pV-C25. The cDNA clones contain 38 nucleotides (nt) of the 5'-untranslated region, 1398 nt of the coding region and 7 nt of the 3'-untranslated region. Comparing the mouse sequence with the published sequence for vimentin from hamster, human and chicken, we find shared identities of 99, 97 and 87%, respectively. Since the cDNA clones have been isolated from a basement membrane tumor (EHS) cDNA library, we measured the vimentin mRNA production in EHS tumor cells in culture, and found that this mRNA is half as abundant as mRNA for type-IV mRNA.     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

Analysis and in situ detection of cholecalcin messenger RNA (9000 Mr CaBP) in the uterus of the pregnant rat

Summary The molecular cloning of a cDNA fragment synthesised from rat duodenal mRNA coding for cholecalcin (calbindin), a 9000 Mr vitamin D-induced calcium-binding protein (CaBP), has been previously described. DNA/RNA hybridisation assays have been used to examine CaBP mRNA production in the uterine horns and duodena of pregnant (21 day) rats using the cloned CaBP cDNA. Northern hybridisation studies showed that the ³²P cDNA sequence hybridised to a single 500–600 nucleotide species in both the uterus and the duodenum, thus demonstrating identical CaBP mRNA processing in both tissues. Dot blot hybridisation studies showed that the CaBP mRNA concentration was greatest in the duodenum while that of the uterine horns was about 10% of the duodenal level. The observed differences in CaBP mRNA levels correlate well with the in vivo CaBP concentrations. In situ hybridisation histochemistry using ³H cDNA revealed that CaBP mRNA visualised by silver grains was found in all the parts of the endometrium and the myometrium. However, CaBP mRNA was more concentrated in the outer and inner muscular fibres and in the luminal cells of the endometrium than in the stroma cells. These results demonstrate that the CaBP gene is expressed in specific cells of the rat uterus.     

Human gamma-glutamyl transpeptidase cDNA: comparison of hepatoma and kidney mRNA in the human and rat

gamma-Glutamyl transpeptidase (GGT) is a glutathione-metabolizing enzyme that has been extensively studied in relation to hepatocarcinogenesis. Using a cDNA for rat kidney GGT as a probe, we have isolated a full-length cDNA for human GGT from a hepatoma cell-line library. Nucleotide sequence analysis of the clone revealed a 2326-bp insert that includes a 5'-untranslated region of 487 nucleotides (nt), an open reading frame (ORF) of 1707 nt, and a 3'-untranslated region of 132 nt. The ORF encodes a protein with an amino acid sequence that is highly similar to that of the rat GGT precursor peptide, with an overall identity of 79%. The cDNA clone was used to probe Northern blots of hepatoma and kidney RNA from both human and rat. In both species, the GGT mRNA is longer in hepatoma than in kidney. In addition, the human mRNAs were longer than their counterparts in the rat. None of three human hepatocellular carcinomas examined showed a marked elevation in GGT mRNA levels relative to surrounding liver tissue.