Human TOP3: a single-copy gene encoding DNA topoisomerase III.

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.     

Synthesis of a gene encoding parathyroid hormone-like protein-(1-141): purification and biological characterization of the expressed protein

PTH-like proteins (PTHLP), which are associated with humoral hypercalcemia of malignancy, have recently been purified. Isolation of their corresponding cDNAs has revealed that they are derived from a single gene. In this report a synthetic gene encoding PTHLP-(1-141), a 141-amino acid protein corresponding to the most abundant PTHLP cDNA detected in human tumors, was expressed in bacteria and purified to homogeneity. Recombinant (r) PTHLP-(1-141) migrates with an aberrantly high mol wt on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, presumably as a result of its unusually basic pI. rPTHLP-(1-141), like PTH, induced hypercalcemia in rats, caused release of 45Ca from fetal rat bones, and stimulated the synthesis of cAMP by rat osteosarcoma cells and canine renal membrane preparations. A comparison of the abilities of rPTHLP-(1-141) and bovine PTH-(1-34) to stimulate cAMP synthesis indicated rPTHLP-(1-141) to be 5-fold more potent in the osteosarcoma assay, while nearly 30-fold less active in the renal membrane adenylate cyclase assay. Although 100-fold less potent than bovine PTH-(1-34) in promoting bone resorption, rPTHLP-(1-141) was a potent calcemic factor in vivo, inducing a rise in serum calcium from 10.4 to 14.5 mg/dl when infused into rats at 1.3 micrograms/h. These results support previous assumptions that PTHLP is the humoral factor responsible for humoral hypercalcemia of malignancy. In addition, they suggest substantial differences between PTHLP and PTH in the regulation of calcium homeostasis.     

Structure of the human gene and two rat cDNAs encoding the alpha chain of GTP-binding regulatory protein Go: two different mRNAs are generated by alternative splicing.

Go is a specific class ("other") of signal-transducing heterotrimeric GTP-binding proteins (G proteins) that is expressed in high levels in mammalian brain. We have cloned two different rat cDNAs encoding the alpha subunit of Go (Go alpha-1 and Go alpha-2) and a human Go alpha chromosomal gene. The human Go alpha gene spans more than 100 kilobases and contains 11 exons, including one noncoding exon in the 3' flanking region. The 5' flanking region is highly G + C-rich and contains five G.C boxes (Sp1 binding sites) but no TATA box. Exons 7 and 8 coding for amino acid residues 242-354 of Go alpha protein are duplicated (referred to as exons 7A, 7B, 8A, and 8B). It was found that exons 7A and 8A code for Go alpha-1, and 7B and 8B code for Go alpha-2. This indicates that two different Go alpha mRNAs may be generated by alternative splicing of a single Go alpha gene. The splice sites of the Go alpha-1 and Go alpha-2 genes are completely identical with those encoding human inhibitory G protein alpha subunits Gi2 alpha and Gi3 alpha [Itoh, H., Toyama, R., Kozasa, T., Tsukamoto, T., Matsuoka, M. & Kaziro, Y. (1988) J. Biol. Chem. 263, 6656-6664] and also transducin G protein alpha subunit Gt1 alpha [Raport, C. J., Dere, B. & Hurley, J. (1989) J. Biol. Chem. 264, 7122-7128]. Sequence homology and conservation of the exon-intron organization indicate that the genes coding for Go alpha, Gi2 alpha, Gi3 alpha, Gt1 alpha, and probably Gi1 alpha may be evolved from a common progenitor. Like Go alpha-1, Go alpha-2 is expressed mainly in brain.     

B-cell variant of mouse T200 (Ly-5): evidence for alternative mRNA splicing.

The complete cDNA sequence of mouse T200 glycoprotein from the pre-B-cell line 70Z/3 has been determined. The deduced protein sequence differs from that previously reported for a T-cell form of the molecule [Saga, Y., Tung, J.-S., Shen, F.-W. & Boyse, E. A. (1986) Proc. Natl. Acad. Sci. USA 83, 6940-6944] by the insertion of 139 amino acid residues in the amino-terminal region of the molecule. RNA transfer blotting using a cDNA probe encoding this sequence established that the predominant T200 mRNA species from B cells and cytotoxic T-cell clones but not T-helper cell clones or thymocytes contain all or part of the insert. Overlapping genomic clones of murine T200 were isolated and analysis of the intron-exon structure at the 5' end of the gene provides evidence that variants of T200 glycoprotein are generated by alternative mRNA splicing.     

Messenger ribonucleic acid from tumors associated with humoral hypercalcemia of malignancy directs the synthesis of a secretory parathyroid hormone-like peptide

Previous studies have provided evidence that tumors associated with humoral hypercalcemia of malignancy produce a factor that shares certain biological properties with, but is not, native PTH. In the present study, media from Xenopus oocytes microinjected with polyadenylated RNA prepared from three human or animal tumors associated with humoral hypercalcemia of malignancy are shown to have activity in the cytochemical bioassay for PTH. This activity parallels that of the PTH standard in the assay and is completely inhibited by the PTH analog [Nle8,Nle18,Tyr34]bovine PTH-(3-34)NH2. Media from oocytes injected with mRNA from control tumors contain no such activity. The mRNA encoding this activity in one of the animal tumors has been enriched approximately 10-fold by preparative polyacrylamide gel electrophoresis. These results demonstrate that tumors associated with humoral hypercalcemia of malignancy have the capacity for directing the synthesis of a secretory protein that fulfills certain criteria for being the humoral mediator in question. These techniques may provide the basis for molecular cloning of this factor.     

Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach.     

Identification of an additional member of the protein-tyrosine-phosphatase family: evidence for alternative splicing in the tyrosine phosphatase domain.

Protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48) have been implicated in the regulation of cell growth; however, to date few tyrosine phosphatases have been characterized. To identify additional family members, the cDNA for the human tyrosine phosphatase leukocyte common antigen (LCA; CD45) was used to screen, under low stringency, a mouse pre-B-cell cDNA library. Two cDNA clones were isolated and sequence analysis predicts a protein sequence of 793 amino acids. We have named the molecule LRP (LCA-related phosphatase). RNA transfer analysis indicates that the cDNAs were derived from a 3.2-kilobase mRNA. The LRP mRNA is transcribed in a wide variety of tissues. The predicted protein structure can be divided into the following structural features: a short 19-amino acid leader sequence, an exterior domain of 123 amino acids that is predicted to be highly glycosylated, a 24-amino acid membrane-spanning region, and a 627-amino acid cytoplasmic region. The cytoplasmic region contains two approximately 260-amino acid domains, each with homology to the tyrosine phosphatase family. One of the cDNA clones differed in that it had a 108-base-pair insertion that, while preserving the reading frame, would disrupt the first protein-tyrosine-phosphatase domain. Analysis of genomic DNA indicates that the insertion is due to an alternatively spliced exon. LRP appears to be evolutionarily conserved as a putative homologue has been identified in the invertebrate Styela plicata.     

Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing.

Rous sarcoma virus was shown to induce in chicken embryo fibroblasts (CEF) a 4.1-kilobase mRNA (designated CEF-147) encoding a 603-amino acid protein. Analysis of the protein sequence showed that it shared 59% amino acid identity with sheep prostaglandin G/H synthase, the enzyme that catalyzes the rate-limiting steps in the production of prostaglandins. Significant differences, at both the protein and mRNA levels, existed between the src oncogene product-inducible prostaglandin synthase and the protein isolated and cloned from sheep seminal vesicle, suggesting that the src-inducible prostaglandin synthase may be a new form of the enzyme. A distinguishing feature of src-inducible prostaglandin synthase mRNA is its low abundance in nonproliferating chicken embryo fibroblasts and its relatively high abundance in src-transformed cells. Additionally, the majority of the src-inducible prostaglandin synthase RNA present in nonproliferating cells was found to be nonfunctional because of the presence of an unspliced intron that separated the signal peptide from the remainder of the protein. Upon mitogenic stimulation, this intron was removed, resulting in the induction of fully-spliced CEF-147 mRNA.     

A muscle-type tropomyosin in human fibroblasts: evidence for expression by an alternative RNA splicing mechanism.

We have isolated a cDNA clone from a human fibroblast cDNA library that contains the entire protein-coding region of a 1.1-kilobase mRNA. This mRNA encodes a 284-amino acid tropomyosin, the primary structure of which most closely resembles smooth muscle tropomyosin. Thus, the expression of both 284-amino acid muscle-type and 247-amino acid non-muscle-type tropomyosins appears to be a normal feature of human non-muscle cells. We also present evidence to suggest that this cytoskeletal tropomyosin and a human skeletal muscle beta-tropomyosin are derived from a common structural gene by an alternative RNA splicing mechanism.     

Bioactivity of parathyroid hormone and parathyroid hormone-like peptide: agonist and antagonist activities of amino-terminal fragments as assessed by the cytochemical bioassay and in situ biochemistry

Parathyroid hormone-like peptide (PLP) is elaborated from certain tumors and is thought to play a role in the etiology of humoral hypercalcemia of malignancy. The amino-terminal portion of this peptide has a sequence homology with parathyroid hormone PTH. We have compared the agonist potency of the synthetic human amino-terminal 1-34 peptide [human (h)PLP-(1-34)] with that of intact PTH and its amino terminal fragment [hPTH-(1-34)] in the renal and metatarsal cytochemical bioassays (CBA). Furthermore, the antagonist activity of the truncated amino terminal molecule [hPLP-(3-34)] has been compared to that of [Norleu8.18,Tyr34]bovine PTH-(3-34)NH2, and we have also tested their ability to stimulate enzyme activities thought to be associated with bone formation and resorption. In the renal CBA, both PLP-(1-34) and hPTH-(1-34) were equipotent with intact hPTH. In the metatarsal CBA, although the two amino-terminal peptides were equipotent, they elicited an earlier response than the intact PTH molecule. In both assay systems the truncated PLP analog [hPLP-(3-34)] was a more potent antagonist of both PTH and PLP activity than was [Norleu8.18,Tyr34]bovine PTH-(1-34)NH2. In acute studies, hPLP-(1-34) and hPTH-(1-34) stimulated alkaline phosphatase and glucose 6-phosphate dehydrogenase activity in osteoblasts to a similar extent, and both peptides stimulated tartrate-resistant acid phosphatase and succinate dehydrogenase activity in osteoclasts. Longer exposure to the peptides resulted in stimulation of enzyme activity in osteoclasts but not osteoblasts, although there was no difference in potency between the two molecules.     

Structure and expression of the gene encoding the vasoactive intestinal peptide precursor.

The gene encoding the human vasoactive intestinal peptide (VIP) and the histidine-methionine amide (PHM-27) peptide hormone was isolated from lambda phage libraries. The human gene was found to be composed of seven exons spanning approximately 9 kilobase pairs. The first exon codes for an untranslated leader sequence, and the second exon codes for a putative signal peptide. DNA sequences coding for the VIP and PHM-27 hormones are located in two different exons. Southern blot analysis with genomic DNA suggested that a single copy of the VIP/PHM-27 gene is present in the human haploid genome. The expression of VIP/PHM-27 precursor mRNA in various tissues in the rat was analyzed by RNA gel blot hybridization. In the organs examined, expression was only detected in the brain and duodenum. RNA isolated from various regions of the rat brain--including the cortex, hypothalamus, and hippocampus--hybridized to both VIP- and PHM-27-specific probes. The same pattern of hybridization was found when VIP- and PHM-27-specific probes were used, suggesting that possible differences in the localization of VIP and PHM-27 peptides between different brain regions cannot be accounted for by differential RNA processing.     

Duck lens epsilon-crystallin and lactate dehydrogenase B4 are identical: a single-copy gene product with two distinct functions.

To investigate whether or not duck lens epsilon-crystallin and duck heart lactate dehydrogenase (LDH) B4 are the product of the same gene, we have isolated and sequenced cDNA clones of duck epsilon-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens.