Judy Miller

Human and canine gene homologs of porcine brain natriuretic peptide

A cDNA clone encoding porcine brain natriuretic peptide (BNP) precursor was used to probe a canine genomic DNA library for homologous sequences. A unique clone was obtained (D1) which encoded a peptide homologous to porcine BNP. A 4.0 kB Hind III fragment of D1 containing the entire BNP precursor coding region was hybridized against blots of EcoRI digested human genomic DNA. A 7.0 kB hybridizing band was observed, which was subsequently subcloned and sequenced. This human clone, H1, also contained a sequence homologous to porcine BNP precursor. Transcription of the human BNP gene was confirmed by detection of BNP-specific sequences amplified from human atrial RNA by polymerase chain reaction. These results suggest that both BNP and ANP are present and function in a wide variety of mammalian species.

The primary structure of coho salmon growth hormone and its cDNA

Total RNA was extracted from coho salmon growth hormone (sGH) cell regions and used to synthesize double-stranded cDNA, which was inserted into a plasmid vector and used to transform Escherichia coli HB101. The total RNA was also separated according to size by electrophoresis on agarose gels and the fraction that directed the cell-free synthesis of protein in the size range of GHs of other species was isolated and used to screen the transformed colonies of E. coli. A clone containing the putative sGH cDNA was identified and its nucleotide sequence was determined. To verify that the cDNA was that of sGH, the GH cell region of coho pituitary glands was incubated in organ culture. The secreted GH was purified by HPLC and the sequence of its 42 amino-terminal amino acids was determined. Comparison of this sequence with the amino acid sequence derived from the cDNA showed that it encoded sGH. Medium containing the presumptive sGH as the only prominent protein was active in a GH radioreceptor assay that involved labeled bovine GH and pregnant mouse liver membranes: the sGH was approximately 10% as active as the bGH standard. RNA blotting analysis showed that sGH was the major species of RNA produced by the GH cell region of the salmon pituitary. The mRNA of sGH differed from those of human, rat, and bovine GH in that its 3-untranslated region was unusually large (about 500 nucleotides) but the coding region showed significant homology with mammalian GHs and resembled them in having a strong (78%) preference for G and C in the third positions of the codons. The amino acid sequence of sGH showed 32-34% and 19-22% identical homology with mammalian GHs and prolactins, respectively. Several conserved regions between sGH and mammalian GH and PRL molecules were also revealed that could indicate conservation of structurally and/or functionally important domains. Hydropathy analysis disclosed that although sGH and the GH of a representative mammal (pig) had similar profiles in some regions, the sGH was overall more hydrophobic than the pig (p) GH. Similarities and differences, were also noted in the predicted secondary structure of sGH and pGH.

Cloning of the fusion gene of rinderpest virus: Comparative sequence analysis with other morbilliviruses

We have cloned the cDNA of the fusion (F) gene of the virulent (Kabete O) strain of rinderpest virus and provided a comparative analysis of its sequence with that of the F genes of measles and distemper viruses. The gene has an open reading frame of 2241 nucleotides with two potential initiation codons in-frame. Use of the first ATG would produce a polypeptide 747 amino acids long with a calculated molecular weight of 81,068. However, we suggest that the second ATG is used to generate the Fo protein, which is 546 amino acids long with a calculated molecular weight of 58,754. During maturation, the cleavage of F0 gives rise to the functional F1 and F2 polypeptides. The F1 polypeptide is 438 amino acids long and has a calculated molecular weight of 46,791, with a single (potential) glycosylation site in its cytoplasmic domain. The F2 polypeptide, probably 89 amino acids long after the signal sequence is cleaved, is estimated to be 9,800 Da and has three potential glycosylation sites. There is a divergence of 18.7% in amino acid sequences between rinderpest and measles virus F0 polypeptides; between distemper and rinderpest viruses the divergence is 31.8%. No significant homology in nucleotide sequences of rinderpest DNA to measles or distemper DNA was found in the 5 and 3 untranslated regions.

Structural analysis of the gene for human acidic fibroblast growth factor

Genomic clones derived from the gene for human acidic fibroblast growth factor (aFGF) have been isolated. Nucleotide sequence analysis of these clones revealed that the coding region of the human aFGF gene is interrupted by two introns, located at precisely homologous locations to introns in four other members of the FGF gene family, strongly indicating a common evolutionary origin for these genes. Northern blot analyses of the multiple aFGF transcripts found in serum-stimulated human foreskin fibroblasts indicated that the aFGF gene also contains a third intron, lying in the 5 untranslated region.