Complementary DNA cloning of a protein highly homologous to mammalian sarcoplasmic reticulum Ca-ATPase from the crustacean Artemia

Complementary DNA clones coding for an Artemia ATPase have been isolated using an oligonucleotide probe for a region highly conserved between P-type ATPases. The nucleotide sequence of three overlapping clones, 3309 base-pairs, has been established. This sequence includes 78 nucleotides of 5' untranslated sequence, an open reading frame of 3009 nucleotides and 222 nucleotides of 3' untranslated sequences. The amino acid sequence predicted for the coding region is 71% similar to that of slow and fast twitch rabbit muscle sarcoplasmic reticulum Ca-ATPases. The homology is specially high in some regions of the protein that include the previously described regions that are similar between all known P-type ATPases, as well as transmembrane domains and intra- and extracellular domains adjacent to the membrane that are not conserved in P-type ATPases but have been proposed to be involved in calcium binding and transport in rabbit sarcoplasmic reticulum Ca-ATPases. Probes of this likely sarcoplasmic reticulum Ca-ATPase hybridize to two mRNAs of 5200 and 4500 bases. Although both mRNAs are already present in cryptobiotic embryos, the levels of the 5200 base mRNA decrease after development is reassumed, being undetectable after hatching of the nauplii. The levels of the 4500 base mRNA increase during development; maximal levels are reached by ten hours and are maintained at later stages of development.     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.     

cDNA cloning, functional expression, and mRNA tissue distribution of a third organellar Ca2+ pump

We describe the characterization of a rat kidney cDNA that encodes a novel Ca2+-transporting ATPase. The cDNA, termed RK 8-13, was isolated previously using an oligonucleotide hybridization probe corresponding to part of the ATP binding site of the sarcoplasmic reticulum Ca-ATPases (Gunteski-Hamblin, A.-M., Greeb, J., and Shull, G. E. (1988) J. Biol. Chem. 263, 15032-15040). The complete nucleotide sequence of the 4.5-kilobase cDNA has been determined, and the primary structure of the protein has been deduced. The enzyme consists of 999 amino acids, has an Mr of 109,223, and contains all of the conserved domains found in transport ATPases of the E1-E2 class. It exhibits 75-77% amino acid identity with the fast-twitch and slow-twitch/cardiac isoforms of the sarcoplasmic reticulum Ca-ATPase, and the hydropathy plots of the three enzymes are virtually identical. High levels of ATP-dependent Ca2+ uptake were demonstrated in microsomes of COS-1 cells that had been transfected with a construct consisting of the entire coding sequence of the cDNA in the expression vector p91023(B). Northern blot analyses of poly(A)+ RNA revealed that the mRNA for this protein is expressed in heart, skeletal muscle, uterus, brain, lung, liver, kidney, testes, small intestine, large intestine, and pancreas. These data show that the enzyme is a Ca2+-transporting ATPase and that its mRNA is expressed in a broad variety of both muscle and non-muscle tissues.     

Expression of the pregnancy-specific beta 1-glycoprotein genes in human testis

Northern blot analysis with placental pregnancy-specific beta 1-glycoprotein (SP1) cDNA probe showed the presence of SP1 mRNAs in human testis. Presence of translational products of the mRNAs was demonstrated by Western blot analysis with anti-human SP1 antibodies albeit difference in mobilities between the testis and placental proteins was apparent. Screening of human testis cDNA library with placental SP1 probe yielded 4 groups of positive clones. Two groups were identical to human placental SP1 cDNAs previously reported. The other 2 groups consisted of cDNA of incompletely processed mRNAs. These 2 groups were present in high abundance. Sequence analysis suggested that the cDNAs were products of different genes.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Molecular cloning and chromosomal localization of a sarco/endoplasmic reticulum-type Ca2(+)-ATPase of Drosophila melanogaster

We report here the characterization of a Drosophila melanogaster cDNA that encodes a sarco/endoplasmic reticulum-type Ca2(+)-ATPase. Previously we amplified the phosphorylation site - FITC-binding site fragment of this cDNA by Polymerase Chain Reaction utilizing homology primers (Váradi, A., Gilmore-Hebert, M. and Benz, E. J. Jr. (1989) FEBS Letters 258 203-207) and in the present work we used this fragment as probe for isolation of a cDNA clone with the intire coding region. The isolated 3.3 kb clone has been sequenced and the primary structure of the encoded protein has been deduced. It consists of 1002 amino acids with all the characteristic features of the P-type ATPases. It shows 67-71% amino acid identity and an apparently identical hydropathy profile with the mammalian sarco/endoplasmic reticulum-type Ca2(+)-ATPases. On basis of sequence similarity we suggest that the first transmembrane segment of sarco/endoplasmic reticulum type Ca2(+)-ATPases may be responsible for targeting of these transport proteins into the organellar membrane. The gene of this sarco/endoplasmic reticulum-type Ca2(+)-ATPase has been mapped on the right arm of chromosome 2, in section 60A.     

Cloning and expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine heart

Distinct 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) cDNAs were cloned from bovine heart, showing that PFK-2/FBPase-2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000-M_r isozyme. In B2 mRNA, exon 15 encodes four more residues than in Bl. In B4 mRNA, exon 15 encodes six more residues than in B1, butexon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000-M_r isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK-2 activity.     

The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase.

The mgtB locus codes for one of three distinct Mg2+ transport systems of Salmonella typhimurium. The system encoded by the mgtB locus mediates Mg2+ influx only. The nucleotide sequence of a 4.6-kilobase fragment of DNA carrying mgtB has been determined. Two open reading frames were apparent. The most 5' (mgtC) could encode a hydrophobic protein of up to 25 kDa depending on which translation starts are used. A plasmid carrying this region downstream from a phage T7 promoter expresses a 22.5-kDa protein. The second open reading frame encoded a 101-kDa polypeptide (MgtB) consistent with our previous observation that a plasmid carrying the mgtB locus expresses a 102-kDa protein in maxicells. Insertions into either open reading frame abolished the ability of the plasmid to relieve the requirement for added Mg2+ and to restore Mg2+ uptake to a Mg2+ transport-deficient strain of S. typhimurium. The predicted amino acid sequence of MgtC showed no similarity to any other known protein. In contrast, the predicted sequence of MgtB indicated that it is a member of the family of cation transport P-type ATPases. Strikingly, however, MgtB was significantly more similar to eukaryotic Ca2(+)-ATPases than to prokaryotic P-type ATPases or other classes of eukaryotic P-type ATPases such as the Na+,K(+)-ATPase. MgtB is most closely related to Ca2(+)-ATPases of mammalian sarcoplasmic reticulum and yeast. A number of features of the Ca2(+)-ATPases thought to be important for cation transduction across the membrane are present in MgtB but not in other prokaryotic members of this enzyme family. Unlike the Ca2(+)-ATPases, however, which mediate efflux of cation from the cytosol, MgtB mediates influx of cation into the cytosol.     

Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B).

Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (beta 1) encodes a 170 amino acid protein (M(r) approximately 19.3 kDa), whereas that for the testis isoform (beta 2) contains 179 residues (M(r) approximately 20.7 kDa); these two sequences show approximately 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the beta 2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (alpha 3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.     

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.     

Epstein-Barr virus mRNAs produced by alternative splicing.

The structure of Epstein-Barr virus mRNAs transcribed in B95-8 cells has been studied by cDNA cloning and sequencing. We present here the analysis of four cDNAs. The corresponding mRNAs are probably transcribed from a single promoter located in the US region. They are produced by alternative splicing of exons transcribed from the US, IR and UL regions. The exons are spread over 100 kbp. The exons from the IR region constitute a unit which is repeated several times. The cDNAs share the exons from the US and IR regions. Some of the cDNAs also share some of the exons from the UL region. Each cDNA contains a long open reading frame or the 5' end of a long open reading frame which ends several hundred nucleotides downstream on the viral genome. The 5' untranslated regions are unusually long. Three mRNA species differing in their 5' untranslated regions may encode for the nuclear antigen EBNA-1. The other mRNAs encode for polypeptides which may not have any common region.     

Molecular cloning and analysis of DNA complementary to three mouse Mr = 68,000 heat shock protein mRNAs

The construction and isolation of three recombinant DNAs complementary to different mouse L-cell Mr = 68,000 heat shock protein (hsp68) mRNAs is described. cDNA libraries derived from heat-shocked mouse L-cell poly(A)+ RNA by the vector-linked primer strategy of cDNA synthesis and cloning of Okayama and Berg (Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) were screened first with a Drosophila hsp70 heterologous probe and subsequently with a cDNA probe isolated from the first screening. Positive clones were assigned to one of three sets based on their restriction map, and the largest member of each group was chosen for further analysis. All three cDNAs hybrid-select mRNA for the mouse major heat shock protein (hsp68) as assayed by in vitro translation and hybridize preferentially to two heat shock-induced hsp68 mRNAs on Northern blots. The coding regions of the cDNAs are almost identical and closely resemble other HSP70 genes but the 3' untranslated regions diverge considerably. Differences in the lengths of the untranslated regions are responsible for the two different sized induced hsp68 mRNAs in mouse L-cells. The physical maps of these cDNA clones and the limited number of mouse genomic DNA fragments detected on Southern blots suggest that there are at least three closely related heat shock-inducible members of the mouse HSP70 gene family. None of the cloned cDNAs are derived from the two related cognate genes known to be present in the mouse genome.