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PubMed1992

MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly.

The MAK3 gene is necessary for propagation of the L-A double-stranded RNA virus of Saccharomyces cerevisiae. MAK3 encodes a protein with substantial homology to the Escherichia coli rimI N-acetyltransferase that acetylates the NH2 terminus of ribosomal protein S18, and shares consensus sequences with a group of N-acetyltransferases. The NH2 terminus of the viral major coat protein encoded by L-A is normally blocked, but we find that it is unblocked in a mak3-1 mutant. L-A virus-encoded proteins produced from a cDNA clone of L-A can encapsidate the L-A (+)-strands in a wild-type host, but not in a mak3-1 mutant strain. The amount of major coat protein found in the particle fraction is reduced greater than 100-fold, and the amount in the total cell extract is reduced 5-10-fold. A modified beta-galactosidase, having as its NH2-terminal the NH2-terminal 13 residues of the L-A-encoded major coat protein, is blocked in a wild-type host, but not in a mak3-1 host. We propose that MAK3 encodes an N-acetyltransferase whose modification of the L-A major coat protein NH2 terminus is essential for viral assembly, and that unassembled coat protein is unstable.

Acetylation↗
PubMed2003

Aberrant promoter methylation and silencing of laminin-5-encoding genes in breast carcinoma.

PURPOSE: Down-regulation of Laminin-5 (LN5)-encoding genes (LAMA3, LAMB3, and LAMC2) has been reported in various human cancers. However, the mechanism of inactivation was not clearly understood until recently. In this study, we investigated the loss of expression of three LN5-encoding genes in breast cancer cell lines and elucidated the mechanism of silencing of the genes in breast cancer cell lines and tumors. EXPERIMENTAL DESIGN: We examined the expression of the three LN5-encoding genes by reverse transcription-PCR in breast cancer cell lines (n = 20). To elucidate the mechanism of silencing, we treated expression negative cell lines (n = 5) with a demethylating agent and examined restoration of expression by reverse transcription-PCR. By using methylation-specific primers designed by us, we validated the methylation status of the promoter regions in breast cancer cell lines using methylation-specific PCR. We additionally studied the methylation patterns in primary breast tumors (n = 74) and correlated the data with clinical parameters. RESULTS: We observed varied losses of expression (10-55%) of LN5-encoding genes in breast cancer cell lines. Expression of one or more genes was lost in 65% of breast cancer cell lines. Treatment of expression negative cell lines with demethylating agent restored expression in all cases. Methylation frequencies of LAMA3, LAMB3, and LAMC2 genes in 20 breast cancer cell lines were 40, 5, and 15%, respectively. The concordances between loss of expression and methylation in 20 breast cancer cell lines for the three genes (85-95%) were statistically significant. Nonmalignant breast tissues (n = 30) had very low frequencies of methylation (0-7%). In 74 breast tumors, methylation frequencies LAMA3, LAMB3, and LAMC2 were 44, 4, and 20%, respectively. The differences in methylation frequencies between cell lines and tumors were not statistically significant for all of the three genes. The methylation frequencies of LAMA3 and mean chain methylation index in cell lines and tumors were significantly different from methylation frequencies in nonmalignant tissues, and they were significantly higher in high stage and large size tumors as compared with low-stage and small size tumors. LAMA3 promoter methylation frequency in breast tumors was associated with increased tumor stage (P < 0.001) and tumor size (P < 0.001). CONCLUSIONS: Our results demonstrate epigenetic inactivation of LN5-encoding genes in breast cancers and association of LAMA3 promoter methylation with increased tumor stage and tumor size. Our findings are of biological interest and potentially of clinical importance.

Breast Neoplasms↗
PubMed1992

Analysis of the tissue-specific distribution of mRNAs encoding the plasma membrane calcium-pumping ATPases and characterization of an alternately spliced form of PMCA4 at the cDNA and genomic levels.

The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) mRNAs are encoded on four different genes designated PMCA1-PMCA4. The primary transcripts from some of these genes are known to be alternately spliced in the region encoding the regulatory domains of the enzymes. The known alternately spliced forms of these Ca(2+)-ATPase mRNAs and a new spliced variant of PMCA4 (PMCA4b), presented here, represent at least nine different mRNAs encoding the Ca(2+)-ATPases. In this report, the examination of the tissue-specific distribution of these alternately spliced mRNAs using polymerase chain reaction amplification of cDNA coupled with Southern blotting revealed that each spliced variant had a unique tissue distribution. PMCA1b and PMCA4a were present in all tissues examined. PMCA1a, PMCA1b, and PMCA4b were expressed in excitable tissues, whereas PMCA1d was expressed only in muscle tissues. PMCA2 was found in liver, adrenal gland, spinal cord, and brain. PMCA3a was present in spinal cord, and PMCA3b in thymus, adrenal gland, spinal cord, and brain. The mRNA for a new spliced variant of PMCA4 (PMCA4b) was detected in this study. Complementary DNAs for this isoform were isolated and characterized from human and bovine brain. This alternately spliced form of the PMCA4 mRNA contained an exon inserted at the splice junction immediately following the sequence encoding the calmodulin-binding domain. As has also been shown for PMCA1a, this insertion produced a shift in the reading frame at the 3'-end of the PMCA4 mRNA that yielded a sequence encoding a Ca(2+)-ATPase lacking a large portion of the C-terminal regulatory domain. When the human PMCA4 gene spanning this region of variable exon splicing was sequenced, it confirmed the intron-exon boundaries where alternate splicing occurs to produce PMCA4a and PMCA4b.

Amino Acid Sequence↗
PubMed1992

The gene encoding Escherichia coli acyl carrier protein lies within a cluster of fatty acid biosynthetic genes.

The gene encoding Escherichia coli acyl carrier protein (ACP) has been isolated and sequenced. The ACP gene (called acpP) was located on the genetic map between fabF and fabD which encode two fatty acid biosynthetic enzymes, 3-ketoacyl-ACP synthase II and malonyl CoA-ACP transacylase, respectively. An open reading frame between acpP and fabD encodes a 26.5-kDa protein that has significant sequence identity (greater than 40%) with two acetoacetyl-CoA reductases and thus is believed to encode a 3-ketoacyl-ACP reductase. This gene (called fabG) is cotranscribed with acpP. Thus, the gene encoding ACP, the key carrier protein of fatty acid synthesis, is located within a cluster of fatty acid biosynthetic genes.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed2005

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

Acetyltransferases↗
PubMed1992

Interactions between Yersinia enterocolitica and the host with special reference to virulence plasmid encoded adhesion and humoral immunity.

This thesis is based on 8 publications and a review of the literature. The aim was to summarize present knowledge on molecular components of Yersinia enterocolitica involved in (i) adhesion, with special reference to plasmid encoded factors and (ii) induction of antibodies in the host, and the quantitation of these two phenomena. Adhesion of Y. enterocolitica was quantitatively studied by methods that measured binding to cultured epithelial cells, mucosal constituents immobilized on polystyrene, intestinal tissue, and nonbiological solid surfaces. The chromosomal inv and ail gene products have been shown by others to be independently able to mediate adhesion to and invasion of cultured epithelial cells. However, the Yersinia virulence plasmid, pYV, also encodes for at least one product that may mediate adhesion. It was shown that pYV carrying Y. enterocolitica strains adhered more efficiently than their isogenic pYV cured derivatives to rabbit and human ileal intestinal tissue and to rabbit ileal brush border membrane vesicles (BBVs). Using Y. enterocolitica mutants that were defective for production of the pYV encoded outer membrane protein, YadA, and Escherichia coli strains carrying the cloned yadA gene it was verified that YadA was responsible for the pYV encoded adhesion. The YadA promoted adhesion was found likely to be non-specific and, at least in part, mediated by hydrophobic interaction. YadA, however, also mediated binding to one or more constituents present in intestinal mucus. Such binding led to decreased ability to penetrate a layer of mucus in vitro and subsequent decreased ability to adhere to BBVs. Based upon these results, it remains uncertain whether Y. enterocolitica is able to benefit from the YadA mediated adhesion in the intestinal millieu. In vivo results have suggested that expression of YadA confers on Y. enterocolitica an increased ability to colonize the intestine, but no definitive conclusions have been reached so far. A large number of antigens were identified in Y. enterocolitica by means of crossed immunoelectrophoresis (XIE). Quantitation of serological cross-reactions between chromosome-encoded Y. enterocolitica antigens and antigens from other bacterial species was performed by means of XIE and proved useful for taxonomic purposes. Using XIE it was shown that infection with Y. enterocolitica induced an antibody response against a wide range of antigens. Tube agglutination, enzyme linked immunosorbent assays (ELISAs) using purified lipopolysaccharide (LPS) or formalin-killed whole pYV carrying cells as antigens, and XIE were evaluated for their applicability to detect recent Y. enterocolitica O:3 infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Bacterial↗
PubMed1990

Structure of the gene encoding the human leukocyte adhesion molecule-1 (TQ1, Leu-8) of lymphocytes and neutrophils.

The leukocyte adhesion molecule-1 (LAM-1, TQ1, Leu-8), expressed by human lymphocytes, neutrophils, monocytes, and their precursors, is a member of the selectin family of cellular adhesion/homing receptors which play important roles in leukocyte-endothelial cell interactions. These cell surface molecules contain an amino-terminal lectin-like domain followed by an epidermal growth factor-like domain and a variable number of short consensus repeat sequences similar to those found in C3/C4 binding proteins. In this report, the structure of the lyam-1 gene that encodes the LAM-1 protein was determined by isolating overlapping genomic DNA clones that hybridized with a LAM-1 cDNA probe. The lyam-1 gene spans greater than 30 kilo base pairs of DNA and is composed of at least 10 exons. The 5' end of the LAM-1 mRNA was mapped by primer extension analysis revealing a single initiation region for transcription. Exons II through X contain translated sequences; exon II encodes the translation initiation codon; exon III, the leader peptide; IV, the lectin-like domain; V, the epidermal growth factor-like domain; VI and VII, the short consensus repeat units; exon VIII, the transmembrane region; exon IX encodes seven amino acids containing a potential phosphorylation site; and exon X encodes the five remaining amino acids of the cytoplasmic tail and the long 3' untranslated region. Sequencing of LAM-1 cDNA clones derived from neutrophils revealed that the protein expressed by neutrophils would be identical in sequence with the protein expressed by lymphocytes and cDNAs that would encode different isoforms of LAM-1 protein were not detected. In addition, the level of LAM-1 expression by lymphocytes and neutrophils from two patients with paroxysmal nocturnal hemoglobinuria, a disorder in which linkage of phosphatidylinositol anchors to proteins is defective, was similar to that of normal controls. Therefore, the usage of exons II through X results in the generation of a single major LAM-1 protein product expressed by lymphocytes and neutrophils.

Base Sequence↗
PubMed1991

Characterization of two cDNAs encoding folate-binding proteins from L1210 murine leukemia cells. Increased expression associated with a genomic rearrangement.

L1210 murine leukemic cells grown under conditions of continuous low folate concentrations acquire increased levels of a high affinity/low capacity folate-binding protein (FBP). Using an oligonucleotide probe complementary to the human FBP, we have cloned and sequenced two murine FBP cDNAs isolated from a library constructed using a L1210 subline adapted for growth on low levels of 5-formyltetrahydrofolate. The encoding proteins, designated FBP1 and FBP2, have predicted Mr values of 29,415 and 28,821, respectively. These proteins share significant sequence identity with each other (70%) and with the deduced sequences of the human- and bovine-encoded FBPs (68-79%). Southern blot analysis of the low folate-adapted cell line revealed that, while neither of the two FBP-encoding genes was amplified, the FBP1 genomic locus had undergone rearrangement. On Northern blot analysis, this rearrangement was reflected in the enhanced expression (greater than 100-fold) of a FBP1-specific transcript which was 200 nucleotides shorter than the corresponding L1210 parental transcript. The increased expression of this transcript coincided with the increased expression of a membrane protein (Mr = 38,000) which could be affinity-labeled with a N-hydroxysuccinimide ester of [3H]folate. Accordingly, the FBP1 transcript appears to encode the high affinity/low capacity FBP. Compared to parental L1210 cells, expression of the FBP2-encoding transcript was unchanged in this cell line and, while the exact nature of the protein is unclear, FBP2 may represent a fetal form of the FBP.

Amino Acid Sequence↗
PubMed1990

Characterization of the human gene that encodes the peptide core of secretory granule proteoglycans in promyelocytic leukemia HL-60 cells and analysis of the translated product.

Based upon the deduced amino acid sequence of a cDNA (cDNA-H4) that had been proposed to encode the peptide core of an eosinophil and a HL-60 cell secretory granule proteoglycan, a 16-amino acid peptide was synthesized. This peptide was then used to elicit rabbit antibodies for study of the translation and post-translational modification of this gene product in hematopoietic cells. When HL-60 cells were radiolabeled for 2 min with [35S]methionine, a protein that migrated in a sodium dodecyl sulfate-polyacrylamide electrophoresis gel with a Mr of 20,000 was immunoprecipitated with the IgG fraction of the anti-peptide serum. Kinetic experiments revealed that within 10 min this radiolabeled precursor protein was converted in HL-60 cells into an Mr approximately 150,000 chondroitin sulfate proteoglycan intermediate. After a 20-min to 1-h chase, this [35S]methionine- or [35S]sulfate-labeled proteoglycan intermediate lost its antigenicity, presumably due to proteolysis of its N terminus. A human genomic library was probed under conditions of high stringency with cDNA-H4 to isolate genomic clones that contain the gene that encodes this proteoglycan peptide core. This gene spans approximately 15 kilobases and consists of three exons. The first exon encodes the 5'-untranslated region of the mRNA transcript, as well as the entire 27-amino acid signal peptide of the translated molecule. The second exon encodes a 49-amino acid region of the peptide core, predicted to be the N terminus of the molecule after its proteolytic processing in the endoplasmic reticulum. The third exon encodes the remainder of the molecule, including its glycosaminoglycan attachment, serine-glycine repeat region. As assessed by S1 nuclease mapping and primer extension analysis, the transcription-initiation site in HL-60 cells for this gene resides 53 base pairs upstream from the translation-initiation site.

Amino Acid Sequence↗
PubMed1990

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes.

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.

Amino Acid Sequence↗
PubMed1989

Construction and nucleotide sequence of a cDNA encoding the full-length preprotein for human branched chain acyltransferase.

A cDNA (1.6 kilobases) for branched chain acyltransferase (E2b) isolated from a human liver library encoded only the amino-terminal half of the protein (Hummel, K. B., Litwer, S., Bradford, A. P., Aitken, A., Danner, D. J., and Yeaman, S. J. (1988) J. Biol. Chem. 263, 6165-6168). Here we report the isolation of other cDNAs which encode the carboxyl-terminal half of E2b and the construction of a cDNA which encodes the entire pre-E2b. cDNA from the original clone encoding the leader sequence, lipoate binding domain, and E3 binding domain was ligated to the cDNA from a clone which by restriction maps contained an additional 3' sequence. Both cDNAs used in the construct made a fusion protein in their original phage isolate recognized by antibodies to E2b. The nucleotide sequence of the constructed cDNA was determined, and the 1431 base pairs in the open reading frame encoded a protein of 477 amino acids. In vitro transcription and translation of this cDNA produced a 57-kDa protein recognized by E2b-specific antibodies. Mouse liver mitochondria imported and processed the 57-kDa protein to a 52-kDa antigenic protein which co-migrated with E2b isolated from tissue. Comparing the protein structure of this human pre-E2b protein with that for other acyltransferase proteins showed a similarity in structure throughout all the proteins suggesting evolutionary conservation. Branched chain acyltransferase from Pseudomonas putida showed the most similarity to human E2b.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1989

Cloning and characterization of the mouse gene that encodes the peptide core of secretory granule proteoglycans and expression of this gene in transfected rat-1 fibroblasts.

A mouse liver genomic library was probed with a 450-base pair AccI----3' gene-specific fragment of a mouse bone marrow-derived mast cell proteoglycan cDNA to isolate 15-18-kilobase (kb) genomic clones containing the gene that encodes the peptide core of mouse secretory granule proteoglycans. Based on the nucleotide sequences of its 2.0-3.5-kb subcloned fragments, this mouse gene consists of three exons. The first exon contains 41 base pairs of untranslated nucleotides that are present in the 5' region of the transcript and also encodes the hydrophobic 25-amino acid signal peptide. The second exon encodes a 48-amino acid sequence that would be predicted to be the N terminus of the peptide core after the signal peptide has been removed in the endoplasmic reticulum. The third exon encodes a 79-amino acid sequence that includes the 15 amino acids immediately preceding an alternating serine-glycine 21-amino acid sequence for glycosaminoglycan attachment, and the subsequent C-terminal 43 amino acids; this exon also contains the 424 untranslated nucleotides present in the 3' region of the transcript. Primer extension and S1 nuclease protection analyses were performed to determine the transcription initiation site of the mouse gene. Rat-1 fibroblasts were cotransfected with the selectable marker pSV2 neo and a lambda clone (lambda MG-PG1) to obtain two rat-1 fibroblast cell lines that had the mouse secretory granule proteoglycan gene integrated into their genomes. RNA blot analysis of both cell lines revealed the presence of the 1.0-kb secretory granule proteoglycan peptide core mRNA transcript, indicating that lambda MG-PG1 contained the entire mouse secretory granule proteoglycan peptide core gene including some of the regulatory elements in its promoter region. The gene that encodes the peptide core of mouse secretory granule proteoglycans is the first proteoglycan gene to have its complete exon/intron organization determined and to be transfected and expressed in another cell type.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of a cDNA that encodes the peptide core of the secretory granule proteoglycan of human promyelocytic leukemia HL-60 cells.

A cDNA that encodes the peptide core of the secretory granule proteoglycan of the human promyelocytic leukemic cell line, HL-60, has been isolated and analyzed. When human genomic DNA was digested and probed under conditions of low stringency with a rat cDNA that encodes a Mr = 18,600 serine/glycine-rich proteoglycan peptide core in L2 yolk sac tumor cells (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1321-1325) and basophilic leukemia-1 cells (Avraham, S., Stevens, R. L., Gartner, M. C., Austen, K. F., Lalley, P. A., and Weis, J. H. (1988) J. Biol. Chem. 263, 7292-7296), a number of DNA fragments were identified. A HL-60 cell-derived cDNA library was therefore screened under conditions of low stringency with the rat probe to identify and isolate a human homologue of this rat proteoglycan peptide core. Analysis of the resulting human cDNA clones indicated that the proteoglycan peptide core that is expressed in HL-60 cells is Mr = 17,600 and contains an 18-amino acid glycosaminoglycan attachment region that consists primarily of alternating serin and glycine. Northern blot analysis of total RNA probed with the human cDNA revealed that the major message for this proteoglycan peptide core in HL-60 cells is approximately 1.3 kilobase pairs in size. When a Southern blot of digested human genomic DNA was probed with the human cDNA, three bands of approximately 6, 9, and 12 kilobase pairs were detected. However, when the Southern blot was probed with the XmnI----3' fragment of this human cDNA, one prominent band was detected, indicating that a single gene encodes this protein in the human. Analysis of the DNA from human/mouse and human/hamster somatic cell hybrids probed with the human cDNA demonstrated that the gene that encodes this molecule resides on human chromosome 10. Because the proteoglycans that are present in the secretory granules of different types of rat and mouse mast cells possess small peptide cores that are rich in serine and glycine, we propose that this HL-60 cell-3 derived cDNA encodes the peptide core of the proteoglycan that is expressed in the secretory granules of this human promyelocytic cell.

Amino Acid Sequence↗
PubMed1987

Adaptation of skeletal muscle to increased contractile activity. Expression nuclear genes encoding mitochondrial proteins.

An increase in mitochondrial biogenesis in mammalian cells requires a coordinated increase in the expression of a number of nuclear genes that encode mitochondrial proteins. To examine the regulatory mechanisms involved, we used specific anti-sense RNA probes to estimate the cellular concentrations of mRNA transcripts of two such nuclear genes in rabbit tibialis anterior muscles subjected in vivo to 10-21 days of indirect electrical stimulation. The unstimulated contralateral muscle in the same animals provided a base line for comparison. Change in expression of mitochondrial proteins was assessed in terms of the enzymatic capacity of citrate synthase and cytochrome oxidase, which increased 2.1-fold after 10 days and 5.5- and 4.1-fold, respectively, after 21 days of stimulation. As a proportion of total cellular RNA, messenger RNA encoding subunit beta of F1-ATPase increased 2.2-fold over control levels after 10 days and 2.3-fold after 21 days; mRNA encoding subunit VIC of cytochrome oxidase increased 1.3-fold and 1.9-fold over control levels after stimulation for 10 and 21 days, respectively. These changes were not attributable to nonspecific effects of stimulation on all mRNA transcripts, since aldolase A mRNA decreased to 26% of control levels after 21 days of stimulation. Furthermore, mRNA transcripts from these nuclear genes encoding mitochondrial proteins did not increase to the same extent as mRNA transcripts of mitochondrial genes such as cytochrome b, which increased 5.9-fold after 21 days of stimulation. We conclude that the increase in mitochondrial biogenesis induced by electrical stimulation of skeletal muscle is supported by pretranslational regulation of expression of nuclear genes encoding mitochondrial proteins. There are, however, indications that translational or post-translational regulatory events may also be involved.

Adaptation, Physiological↗
PubMed1989

Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase.

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.

Amino Acid Sequence↗
PubMed1987

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes.

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.

Amino Acid Sequence↗
PubMed1988

Genetics and primary structure of V kappa gene segments encoding antibody to streptococcal group A carbohydrate. Comparison of V kappa gene structure with idiotope expression.

Two gene segments, V kappa-25-39 and V kappa-25-47, that encode antibody to streptococcal group A carbohydrate in A/J mice were found to be more than 95% homologous in nucleotide sequence in both coding and noncoding regions. It was previously shown that V kappa-25-39 encodes immunoglobulins that express the IdX and IdI-1 idiotopes, whereas V kappa-25-47 encodes IdX+, and IdI-1- immunoglobulins. V kappa gene segments that were clearly allelic to V kappa-25-47 are used to encode IdX+, IdI-1- anti-group A carbohydrate antibodies by C.B20 mice and likely by C57BL/6 mice. Murine strains that are deficient in IdI-1 idiotope expression were investigated by Southern blotting with a 5' probe from V kappa-25-39. Two IdI-1-deficient strains, CE/J and C58/J, had a grossly altered V kappa gene segment structure compared with the A/J prototype. In contrast, the IdI-1-deficient strain, C57BL/6, was indistinguishable from A/J with the 5'V kappa-25-39 probe, indicating that more subtle genetic changes account for the loss of IdI-1 expression in C57BL/6 mice. The evolution of V kappa-25-39 and V kappa-25-47 gene segments was deduced by comparison with the homologous V kappa 24B gene segment of Mus pahari. V kappa-25-39 and V kappa-25-47 likely have recently duplicated once in A/J and related strains of laboratory mice and may have duplicated again in CE/J mice. Thus, individual members of the V kappa 24 gene family, to which V kappa-25-39 and V kappa-25-47 belong, are preserved while the number of gene copies expands or contracts. This fact is strong evidence that evolutionary forces have maintained the V kappa 24 gene family, all of which encode antibody specific for carbohydrate found in bacterial pathogens.

Amino Acid Sequence↗
PubMed1988

Phasic alertness and differences in picture encoding speed.

The relation between physical identity and name identity encoding speed was assessed. Correlational analyses indicated that similar processes may be involved in the two types of encoding and, by implication, that differences between mentally retarded and nonretarded subjects' physical identity and name identity encoding speed may result from common factors. Also, we assessed the extent to which differences in preparation for the presentation of external stimuli may account for any observed differences in encoding speed. A difference between retarded and nonretarded subjects in preparation time was apparent; however, this difference did not influence encoding speed.

Adult↗