Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component

Complementary and genomic DNA clones corresponding to the human C-reactive protein (CRP) mRNA and structural gene have been analyzed and compared. Nucleotide sequencing of the coding regions of both cDNA and genomic DNA revealed an additional 19 amino acid peptide not described in the published CRP amino acid sequence. The CRP gene contains a single 278 base pair intron within the codon specifying the third residue of mature CRP. The intron contains a repetitive sequence (GT)15G(GT)3 which is similar to structures capable of adopting the Z-DNA form. A comparison of CRP coding and amino acid sequences with those of serum amyloid P component revealed striking overall homology which was not uniform: a region of limited conservation is bounded by two highly conserved regions.     

Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.

Full length cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rat and man have been isolated and sequenced. Many GAPDH gene-related sequences have been found in both genomes based on genomic blot hybridization analysis. Only one functional gene product is known. Results from genomic library screenings suggest that there are 300-400 copies of these sequences in the rat genome and approximately 100 in the human genome. Some of these related sequences have been shown to be processed pseudogenes. We have isolated several rat cDNA clones corresponding to these pseudogenes indicating that some pseudogenes are transcribed. Rat and human cDNAs are 89% homologous in the coding region, and 76% homologous in the first 100 base pairs of the 3'-noncoding region. Comparison of these two cDNA sequences with those of the chicken, Drosophila and yeast genes allows the analysis of the evolution of the GAPDH genes in detail.     

Expression of atrial natriuretic factor gene in heart ventricular tissue

A novel peptide hormone, atrial natriuretic factor (ANF), was recently isolated and characterized in mammalian atria. This hormone has potent natriuretic, diuretic and vasorelaxant activities. Since ANF bioactivity was initially found in atria but not in ventricles, it was assumed that the ANF gene is specifically expressed in atria. We now report that ANF mRNA is present in ventricular tissue as well as in atria. This is clearly demonstrated by in situ hybridization and by Northern blot analysis. Rat ventricular ANF mRNA concentration is a hundred-fold lower than in atria. As in atria, the 126 amino acids precursor form of ANF is predominant in ventricles and it is present at a thousand-fold lower concentration. The ten-fold discrepancy in the ratio of ANF mRNA to immunoreactivity between atria and ventricles could reflect a higher rate of peptide release in the latter. Thus, ventricular ANF production may be physiologically significant in view of the much larger ventricular mass.     

Thyroid hormone stimulates rat pro-natriodilatin mRNA levels in primary cardiocyte cultures

Pro-natriodilatin (PND) is the precursor for atrial natriuretic peptide (ANP), a hormone which plays an important role in cardiovascular homeostasis. Since the effects of thyroid hormone (T3) on the cardiovascular and renal systems appear to mimic those elicited by ANP, we studied the effect of T3 on PND gene expression using rat neonatal cardiocytes in primary cultures. Treatment of cardiocytes for 48 h with T3 (5 X 10(-9) M) results in a maximal increase in PND mRNA levels; this increase is two fold in atrial and four fold in ventricular cell cultures. These results taken together with a previous report showing decreased plasma ANP in hypothyroid and increased plasma ANP in hyperthyroid rats suggest that at least some of the cardiovascular and renal effects of T3 may be mediated by a T3-dependent increase in PND gene expression.     

Structure and expression of a newt cardio-skeletal myosin gene. Implications for the C value paradox

As part of our studies on the fate of the muscle lineage during amphibian limb regeneration, we have isolated genomic and cDNA sequences from a myosin heavy chain in the newt (Notophthalmus viridescens). Notwithstanding the technical problems inherent in analysing the large newt genome, genomic and cDNA sequences have been isolated and subjected to analysis by restriction mapping. Northern hybridization, Southern hybridization and DNA sequencing. We believe these to be the first single copy newt gene sequences to have been subjected to this type of analysis. The newt gene sequences showed a striking difference from mammalian myosins in both the estimated sizes of the gene and its intervening sequences; these being much larger than in the mammalian models, it is speculated that this could contribute to the exceptional size of the newt genome. By contrast, the coding sequences displayed very high levels of sequence homology to mammalian myosins. In particular, the amino acid sequence of the newt myosin was found to have greatest homology with rat and human myosin isotypes having a similar cardio-skeletal muscle expression pattern. Despite a long evolutionary separation, newt and mammalian cardio-skeletal myosins have remained more similar to each other than have the human or rat cardiac forms to skeletal myosins within their own respective species.     

Three types of rat U1 small nuclear RNA genes with different flanking sequences are induced to express in vivo

There are about 50 copies of U1 RNA genes/pseudogenes in the rat genome. To date, we have isolated so far 25 phage clones carrying a U1 RNA gene/pseudogene from two rat genomic libraries. The 12 clones were selected by hybridization with the U1 RNA coding region under a stringent condition, and were mapped and sequenced. Here, we report three types of U1 RNA genes with different flanking sequences, all of which were shown to be induced to express in vivo by transfection with their polylinker-inserted maxi U1 RNA genes into cultured rat cells. Although these three classes of U1 RNA genes have few homologous flanking sequences, they provide both upstream and downstream of the genes two conserved blocks, which may possibly play an important role in U1 RNA expression.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions

Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen.     

Cloning and expression of the atrial natriuretic factor gene

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone with potent natriuretic, diuretic and vasodilator properties. Isolation and DNA sequence analysis of rat and human cDNA clones revealed that ANF is synthesized from a 126-amino acid precursor which is highly conserved in both species. Southern blot analysis indicated that the ANF gene is present in a single copy per haploid genome. Both human and rat ANF genes were isolated and showed a similar structural organization which consisted of three exons and two introns. The ANF gene was localized to the short arm of human chromosome 1 and mouse chromosome 4. While atria are the major site of expression of the ANF gene in adult heart, other tissues like ventricles, lung, anterior pituitary, hypothalamus and adrenal synthesize ANF albeit to a much lower extent. In ventricles, ANF mRNA levels are 150 times lower than in atria. However, in cardiac hypertrophy or in congestive heart failure, ventricular ANF mRNA and peptide levels are dramatically (100-fold) increased both in animal models and in humans. This suggests that ventricles are a major site of ANF gene expression in certain pathophysiological conditions and that ANF is not an exclusively atrial peptide as was originally thought.