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Biomedical subjects

N B Hecht

Publications and source records attributed to N B Hecht.

At least 127 records · Page 7Linked to original sources
PubMed1983

Haploid accumulation and translational control of phosphoglycerate kinase-2 messenger RNA during mouse spermatogenesis.

The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.

Animals↗
PubMed1983

cDNA clones encoding cytoplasmic poly(A)+ RNAs which first appear at detectable levels in haploid phases of spermatogenesis in the mouse.

We have isolated several cDNA clones encoding cytoplasmic poly(A)+ RNAs which are enriched in postmeiotic (haploid) spermatogenic cells in the mouse. Seventeen of 750 clones from a testis cDNA library hybridized more strongly to 32P-labeled cDNA copied from cytoplasmic poly(A) RNA of round spermatids than pachytene spermatocytes. Northern gel blots demonstrated that these 17 plasmids hybridized to RNA(s) approximately 0.5 kb (1 clone), 0.7 kb (13 clones), 0.8 kb (1 clone), and 0.9 kb (2 clones). Four plasmids hybridizing to RNAs 0.7 and 0.9 kb were further characterized by Northern blots. The levels of hybridization were about 10-fold greater with RNA from round spermatids, elongating spermatids and residual bodies than from pachytene spermatocytes from adult testis. These plasmids did not hybridize with cytoplasmic poly(A)+ RNA from sexually immature testis, adult liver, or brain, larger precursors in adult testis nuclear RNA, total RNA from cultured Sertoli cells, poly(A)- RNA from adult testis or the mouse mitochondrial genome. These results demonstrate that certain poly(A)+ RNAs are abundant in haploid cells but barely or not detectable in meiotic cells suggesting the accumulation of these RNAs in round spermatids requires transcription in haploid cells.

Animals↗
PubMed1983

Gene expression during mammalian spermatogenesis. III. Changes in populations of mRNA during spermiogenesis.

Round spermatids and elongating spermatids were purified from a suspension of mouse testicular cells by sedimentation at unit gravity coupled with density gradient centrifugation through Percoll. Following separation, the two cell types were fractionated into polysomal and non-polysomal compartments. By comparison with round spermatids, elongating spermatids contain about one-half as much cytoplasmic RNA per cell, one sixth as much poly(A)+ RNA per cell and one-half the concentration of poly (A)+ mRNA in their cytoplasm. About two-thirds of the poly(A)+ messenger RNA (mRNA) was in the non-polysomal fraction in both cell types. Polypeptides whose synthesis was directed by cell-free translation of purified mRNA from each cell fraction were analyzed by two-dimensional gel electrophoresis. At the level of detection provided by the electrophoretic methods used, the majority of peptides from the polysomal and non-polysomal compartments for each cell type were similar. However, between the two cell types, approx. 5-10% of the polypeptides in the polysomal and non-polysomal fractions differed markedly in abundance. When the polypeptides encoded by the polysomal and non-polysomal mRNA from round spermatids were compared to the polypeptides encoded in the equivalent fractions from elongating spermatids, a significant reduction in number of polypeptides from elongating spermatids was seen. The presence of specific mRNAs in the non-polysomal fraction of round spermatids and in the polysomal fraction of elongating spermatids suggests that storage of mRNA in the cytoplasm and subsequent utilization provides a source of mRNA for proteins expressed at a time during spermiogenesis when transcription has terminated.

Animals↗
PubMed1983

Gene expression during mammalian spermatogenesis. I. Evidence for stage-specific synthesis of polypeptides in vivo.

Following intratesticular injection of [35S]methionine or [3H]leucine, four testicular cell types (pachytene spermatocytes, round spermatids, elongating spermatids and residual bodies) were purified from mouse testicular cell suspensions by unit gravity sedimentation and equilibrium density gradient centrifugation through Percoll. Measurement of the amount of radiolabeled amino acid incorporated into protein in the testicular cells revealed that for a constant number of cells, pachytene spermatocytes incorporated 5.4 times more isotope than round spermatids, which incorporated 2.4 times more isotope than elongating spermatids. Analysis by two-dimensional gel electrophoresis of the polypeptides synthesized in vivo in the four testicular cell types demonstrated qualitative and quantitative changes in protein synthesis during spermatogenesis. At the level of detection provided by the electrophoretic methods used, pachytene spermatocytes and round spermatids synthesized approximately equivalent numbers of polypeptides while the number of polypeptides synthesized in elongating spermatids and residual bodies was decreased. Quantitative changes for polypeptides ranging in molecular weight from 16,500 to 82,000 were detected during spermatogenesis. For each cell type examined, a minimum of 5% of the polypeptides appear to be either unique or greatly enriched. These studies indicate that the expression of a sizable number of polypeptides is specific to certain stages of spermatogenesis.

Animals↗
PubMed1981

Identification of living spermatogenic cells of the mouse by transillumination-phase contrast microscopic technique for 'in situ' analyses of DNA polymerase activities.

The stages of spermatogenesis can be identified in freshly isolated, unstained adult mouse seminiferous tubules using a transillumination method. Late acrosome- and maturation phase spermatids, arranged in bundles at stages XII-VI give rise to a spotty transillumination pattern. Before spermiation, these cells form a continuous layer on the top of the seminiferous epithelium, recognized by a strong homogeneous central light absorption in the freshly isolated seminiferous tubules at stages VII and VIII. Other stages have a pale light absorption pattern. The accurate determination of the developmental stages of the germ cells was based on the morphology of the developing acrosomic system and of the nuclei of the spermatids, as revealed by phase contrast microscopy. Using this procedure, the activity levels of DNA polymerases alpha and beta have been studied by autoradiography of squash preparations. Using endogenous templates, assay conditions that differentiate between the solubilized DNA polymerases alpha and beta in vitro, were used to distinguish between these activities in situ in different stages of mouse spermatogenesis. Except in very late spermatids shortly before spermiation, DNA polymerases alpha and beta were detectable in all cell types examined. Coinciding with the nuclear protein transitions, elongating spermatids at steps 10-12 and maturation phase spermatids at steps 13-14 showed high DNA polymerase activities. As no replication occurs in these cells, the observations support the view that both DNA polymerases alpha and beta could be involved in repair DNA synthesis.

Animals↗
PubMed1980

Splice point sequence and transcripts of the intervening sequence in the mitochondrial 21S ribosomal RNA gene of yeast.

By S1 nuclease mapping we have located the intervening sequence in the large ribosomal RNA gene of Saccharomyces cerevisiae omega+ strains 570 bp from the 3' end of the rRNA gene. No intervening sequence was detected at this position in S. carlsbergensis, but the sequences of the mature 21S rRNAs of these two strains appear to be identical in this region. By comparing the DNA sequence of the region of the intervening sequence in an omega+ strain with the corresponding sequence in S. carlsbergensis, we have determined the splice points of the 21S rRNA gene. These sequences show no homology with splice points in nuclear and viral genes or with the splice points in the chloroplast 23S rRNA gene of Chlamydomonas. The external borders of the splice points have a complementary sequence in the intervening sequence. The largest transcript hybridizing with the probe of the intervening sequence has a size corresponding to that expected for an rRNA precursor still containing the intervening sequence; the smallest transcript corresponds in size to the intervening sequence itself.

Base Sequence↗
PubMed1979

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.

Animals↗
PubMed1979

Nuclear and mitochondrial DNA-dependent RNA polymerases in bovine spermatozoa.

DNA-dependent RNA polymerases have been solubilized from separated head and tail fractions from normal bovine spermatozoa and from spermatozoa carrying the 'decapitated sperm defect'. When enzyme extracts from separated heads and tails were chromatographed on DEAE-Sephadex, the head fraction was resolved into 2 distinguishable peaks eluting at about 0.11 and 0.15 M-(NH4)2SO4 while the tail fraction yielded 4 distinct peaks eluting at about 0.11, 0.15, 0.255 and 0.35 M-(NH4)2SO4. Results indentical to those observed for sperm tails were obtained with extracts prepared from highly purified mitochondria from bovine or murine heart or liver. Optimization of reaction parameters and inhibitor studies with alpha-amanitin and rifampicin revealed strong similarities between eucaryotic nuclear RNA polymerases 1 and 2 and the 2 RNA polymerases associated with sperm heads. Similar experiments comparing the RNA polymerases from somatic mitochondria and sperm tails suggested the sperm tail enzymes were mitochondrial in origin.

Animals↗
PubMed1977

RNA polymerase activity in bovine spermatozoa.

Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.

Acriflavine↗
PubMed1975

A DNA-directed DNA polymerase from murine liver mitochondria.

A DNA-directed DNA polymerase has been isolated from murine liver mitochondria. The mitochondrial DNA polymerase is distinguishable from other DNA polymerases found in the nucleus and cytosol of murine cells by several enzymatic and physical properties. It is stimulated 5--6-fold by 0.15 M KCl, does not require a sulfhydryl reducing agent for activity, and is inhibited by ethidium bromide or ATP. The enzyme has a sedimentation coefficient of 8.8 S in the presence of up to 0.5 M KCl, a molecular weight of 150--170000, and utilizes natural templates in the following order of preference: activated DNA (100%), single stranded DNA (24%), and native DNA (5%).

Animals↗
PubMed1975

The relationship between two murine DNA-dependent DNA polymerases from the cytosol and the low molecular weight DNA polymerase.

After aqueous subcellular fractionation and partial purification by phosphocellulose chromatography, murine cells are found to contain a low molecular weight DNA-dependent DNA polymerase (beta) in the nuclear fraction and two distinguishable DNA-dependent DNA polymerases (C-I and C-II) in the cytosol. Both C-I and C-II are found in testis, liver, and regenerating liver; the amount of C-I being several fold increased in the regenerating liver and in immature testis. C-I and C-II are distinguishable by the criteria of salt sensitivity, inhibition by single-stranded DNA, elution from phosphocellulose, inhibition by 0.3 mM N-ethylmaleimide, template preference, and sedimentation coefficient. C-II is dissociated by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S while C-I is not dissociated, maintaining its sedimentation coefficient of 7.2 S. Many similar chemical and physical properties of C-II and the low molecular weight nuclear DNA polymerase (beta) suggest that C-II may represent an aggregate state of beta monomers, The size, reaction properties and the increase in enzyme activity under conditions of rapid cellular proliferation suggest C-I is analogous to the alpha DNA polymerase.

Animals↗