Apurinic/apyrimidinic (AP) site recognition by the 5'-dRP/AP lyase in poly(ADP-ribose) polymerase-1 (PARP-1).

The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP-1 was only weakly activated to conduct poly(ADP-ribose) synthesis upon binding to AP site-containing DNA, but was strongly activated for poly(ADP-ribose) synthesis upon strand incision by AP endonuclease 1 (APE1). By virtue of its binding to AP sites, PARP-1 could be poised for its role in base excision repair, pending DNA strand incision by APE1 or the 5'-dRP/AP lyase activity in PARP-1.

Genomic organization and chromosome localization to band 19p13.3 of the human AES gene: gene product exhibits strong similarity to the N-terminal domain of Drosophila enhancer of Split Groucho protein.

The human Amino Enhancer of Split (AES) gene encodes a protein of 197 amino acids exhibiting strong similarity to the N-terminal domain of Drosophila Enhancer of Split Groucho (ESG) protein. The nucleotide sequence of approximately 12 kb from the human AES gene was determined, and its protein-encoding sequence was shown to be interrupted by six introns. The human AES gene was further localized by fluorescence in situ hybridization to chromosome band 19p13.3 near the transcription factor 3 (TCF3) gene.

Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat.

The nucleotide sequences of the cDNAs encoding LDH (EC 1.1.1.27) subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously.

Molecular cloning and expression of cDNAs encoding two isoforms of protein phosphatase 2C beta from mouse testis.

Two isoforms of protein serine/threonine phosphatase were isolated and sequenced from mouse testis. A deletion of 48 nucleotides of PP2C beta 4 cDNA in comparison with PP2C beta 3 cDNA resulted in different COOH-terminal sequences of 12 and 15 amino acids, respectively. These COOH-terminal sequences of PP2C beta 3 and PP2C beta 4 were further found to be different from those of isoforms MPP beta 1 and MPP beta 2 of mouse PP2C beta reported (Terasawa, et al. Arch. Biochem. Biophys. 307: 342-349, 1993). The common sequence of 378 amino acids from these four isoforms of mouse PP2C beta exhibited 95% identity with the corresponding sequence of rat PP2C beta. The mRNAs of approximately 2.0 Kb for PP2C beta 3 and PP2C beta 4 were expressed only in testis, while the mRNAs of 3.3 Kb and 8.5 Kb for MPP beta 1 and MPP beta 2, respectively, were found in somatic tissues.

Molecular cloning and expression of mouse and human cDNA encoding AES and ESG proteins with strong similarity to Drosophila enhancer of split groucho protein.

Mouse and human cDNA encoding AES (amino-terminal enhancer of split) and ESG (enhancer of split groucho) proteins with strong similarity to Drosophila enhancer of split groucho protein were isolated and sequenced. Mouse AES-1 and AES-2 proteins, probably resulting from alternative splicing, contain 202 and 196 amino acids, respectively, while mouse ESG protein consists of 771 amino acids. The amino acid sequences of mouse and human AES proteins were found to exhibit approximately 50% identity to the amino-terminal region of Drosophila groucho, mouse ESG and human transducin-like enhancer of split (TLE) proteins. Mouse AES transcripts of 1.5 kb and 1.2 kb were abundantly expressed in muscle, heart and brain. Human AES transcripts of 1.6 kb and 1.4 kb were predominantly present in muscle, heart and placenta. Mouse ESG (homolog of human TLE 3) transcripts of 3.3 kb and 4.0 kb were found only in testis, while human TLE 1 transcripts of 4.5 kb was more abundant in muscle and placenta compared to heart, brain, lung, liver, kidney and pancreas. Human AES, TLE 1 and TLE 3 genes were mapped to chromosomes 19, 9 and 15, respectively, using human and Chinese hamster hybrid cell lines.

Sequence analysis of mouse cDNAs encoding ribosomal proteins L12 and L18.

Mouse cDNAs encoding ribosomal proteins (r-proteins), L12 and L18, were isolated and their sequences determined. The L12 cDNA was found to contain 639 bp, including a coding sequence of 498 nucleotides (nt), 5' (78 nt) and 3' (45 nt) untranslated regions (UTRs), and a poly(A) tail of 18 nt. The L18 cDNA was shown to consist of 648 bp, including a coding sequence of 567 nt, 5' (26 nt) and 3' (39 nt) UTRs, and a poly(A) tail of 16 nt. The nt sequences of the protein-coding region from the mouse L12 and L18 cDNAs were found to exhibit 96% and 92% identity, respectively, with those of the rat. With the use of mouse L12 and L18 cDNA probes, multiple (at least 10) copies of the L12 and L18 gene families were shown to be present in the mouse and rat genomes. However, there was no sequence heterogeneity detected among seven L18 cDNA clones, indicating that only one copy of the L18 gene-related sequences is functional, and the other copies are presumably nonfunctional pseudogenes. The complete amino acid (aa) sequences of the mouse r-proteins, L12 and L18, were deduced from the nt sequences of their cDNA clones. L12 has 165 aa and a M(r) of 17,790, while L18 has 188 aa and a M(r) of 21,570. The aa sequences of the mouse r-proteins, L12 and L18, exhibit 98% and 94% identity, respectively, to those of rat.

Identification of genes associated with tumor suppression in Syrian hamster embryo cells.

Loss of a tumor-suppressor gene function appears to play a critical role in the multistep process of neoplastic transformation of Syrian hamster embryo (SHE) cells in vitro. Clonal variants of two independent, preneoplastic cell lines have been isolated that have either retained (termed supB+) or lost (termed supB-) the ability to suppress the tumorigenicity of a highly malignant benzo[alpha]pyrene-transformed SHE cell line (BP6T) in cell hybrids. We have pursued several approaches in an attempt to identify genes that are responsible for tumor suppression in these cells. The only consistent differences detected in two-dimensional gel analyses of supB+ and supB- cellular proteins were decreases in the levels of two high molecular weight isoforms of tropomyosin in supB- cells. Differential screening of a supB+ cDNA library for genes that are preferentially expressed in supB+ cells yielded cDNA clones for four genes, i.e., collagen type II, collagen type IX, H19, and a previously unidentified gene (clone 5). Nuclear run-on assays suggested that higher transcription rates were responsible for the increased steady-state levels of some of these transcripts in supB+ cells. DNA sequence comparisons showed that two copies of a 9 bp element, previously identified in each of the mouse H19 enhancers, were also present in the 5' flanking region of the rat type II collagen gene. A transcription factor that controls expression of the collagen and H19 genes through binding to this conserved motif would be an attractive candidate for the supB+ gene or at least a mediator of the supB+ phenotype.

Estrogen-induced expression of mouse lactate dehydrogenase-A gene.

The synthesis of lactate dehydrogenase-A isoenzyme was shown to increase significantly in the uterus of immature mice treated by diethylstilbestrol. The expression of the mouse LDH-A promoter and cat fusion gene in Chinese hamster ovary cells was also induced by 17 beta-estradiol and diethylstilbestrol.

Differential activity and synthesis of lactate dehydrogenase isozymes A (muscle), B (heart), and C (testis) in mouse spermatogenic cells.

Spermatogenic cells isolated from adult and prepubertal mice by unit gravity sedimentation were used to examine enzyme activities and synthesis of the lactate dehydrogenase (LDH) isozymes during spermatogenesis. The synthesis and activity of LDH-C4, the germ cell-specific isozyme, was detected earliest in isolated preleptotene and leptotene/zygotene spermatocytes prior to the mid-pachytene stage of meiosis reported previously. The LDH-C4 isozyme was prominent in pachytene spermatocytes, round spermatids, and condensing spermatids, whereas spermatozoa contained only the LDH-C4 isozyme. In addition, somatic-type LDH isozymes consisting primarily of LDH-B subunits were present in germ cells throughout spermatogenesis. This is in contrast to a previous report that the LDH-B subunit was not synthesized in germ cells. Sertoli cells were further shown to exhibit comparable amounts of five tetrameric LDH isozymes formed by combination of muscle-type LDH-A and heart-type LDH-B subunits.

Expression of the mouse lactate dehydrogenase-A promoter fused with the bacterial gpt gene in Chinese hamster ovary cells.

The promoter region of the cloned mouse lactate dehydrogenase-A gene was fused with the gpt gene of Escherichia coli, and this fusion gene was shown to express in Chinese hamster ovary cells. This result demonstrates that the cloned LDH-A promoter is indeed functional.

Expression of the cDNA encoding for mouse sperm-specific lactate dehydrogenase C in Chinese-hamster ovary cells.

The cloned cDNA encoding for mouse sperm-specific lactate dehydrogenase C (LDH-C) was inserted immediately downstream to the MMTV 5' LTR promoter, and it was shown to synthesize mouse LDH-C polypeptide in Chinese-hamster ovary cells. The mouse LDH-C subunit and the endogenous Chinese-hamster LDH-A subunit formed in vivo a heterotetrameric LDH-A3C1 isoenzyme, and this novel isoenzyme exhibited enzymic activity utilizing lactate as substrate.

Functional expression of the cDNA encoding for human lactate dehydrogenase-A in Chinese hamster ovary cells.

The cloned cDNA encoding for human lactate dehydrogenase-A was inserted immediately downstream to the SV40 early promoter, and it was shown to synthesize the human LDH-A polypeptide in Chinese hamster ovary cells. The human LDH-A subunit and the endogenous Chinese hamster LDH-a subunit formed in vivo a heterotetrameric LDH-a3A1 functional isoenzyme, indicating the conserved tertiary structure of both LDH-A subunits.

Cyclic AMP-induced expression of the mouse lactate dehydrogenase-A promoter-cat fusion gene in Chinese hamster ovary wild-type cells, but not in cAMP-dependent protein kinase mutant cells.

The promoter region of mouse lactate dehydrogenase-A gene was fused with the chloramphenicol acetyltransferase gene of Escherichia coli, and the expression of this fusion gene was induced by cyclic AMP in Chinese hamster ovary wild-type cells, but not in mutants affecting the regulatory or catalytic subunit of cAMP-dependent protein kinase.