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N B Hecht

Publications and source records attributed to N B Hecht.

At least 55 records · Page 3Linked to original sources
PubMed1996

Molecular cloning of mouse somatic and testis-specific H2B histone genes containing a methylated CpG island.

We have isolated a mouse testis-specific H2B histone gene based on the unusual methylation of the CpG island of rat testis-specific H2B gene in somatic tissues. After digestion of genomic DNA with the methylation-sensitive restriction enzyme Hha I, we found that, among 10-20 copies of mouse H2B histone genes, at least three copies are methylated in somatic tissues, but not in testis. Cloning and sequence analysis of two methylated H2B genes revealed that one gene, MTH2B, is strikingly similar to the testis-specific histone H2B (TH2B) gene of rat and the other, psH2B, is a pseudogene of the somatic-type H2B gene. Northern blot analysis revealed that the expression of the MTH2B gene is testis-specific. During spermatogenesis, the MTH2B gene is expressed predominantly in pachytene spermatocytes, as observed in the expression of rat TH2B gene. Interestingly, the MTH2B gene is largely unmethylated in embryonic stem cells, but methylated in F9 embryonal carcinoma cells. The psH2B pseudogene is methylated in somatic tissues and F9 cells, but only partially methylated in embryonic stem cells. Methylation of the psH2B pseudogene seems to be attributed to its location within the context of repetitive sequences including the B1 element. The unmethylation of both H2B histone genes in the testis explains how CpG islands of those histone genes can be maintained during evolution despite heavy methylation in somatic tissues.

Amino Acid Sequence↗
PubMed1995

Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules.

We have previously identified a testicular phosphoprotein that binds to highly conserved sequences (Y and H elements) in the 3' untranslated regions (UTRs) of testicular mRNAs and suppresses in vitro translation of mRNA constructs that contain these sequences. This protein, testis/brain RNA-binding protein (TB-RBP) also is abundant in brain and binds to brain mRNAs whose 3' UTRs contain similar sequences. Here we show that TB-RBP binds specific mRNAs to microtubules (MTs) in vitro. When TB-RBP is added to MTs reassembled from either crude brain extracts or from purified tubulin, most of the TB-RBP binds to MTs. The association of TB-RBP with MTs requires the assembly of MTs and is diminished by colcemid, cytochalasin D, and high levels of salt. Transcripts from the 3' UTRs of three mRNAs that contain the conserved sequence elements (transcripts for protamine 2, tau protein, and myelin basic protein) are linked by TB-RBP to MTs, whereas transcripts that lack the conserved sequences do not bind TB-RBP. We conclude that TB-RBP serves as an attachment protein for the MT association of specific mRNAs. Considering its ability to arrest translation in vitro, we propose that TB-RBP functions in the storage and transportation of mRNAs to specific intracellular sites where they are translated.

Animals↗
PubMed1995

In male mouse germ cells, copper-zinc superoxide dismutase utilizes alternative promoters that produce multiple transcripts with different translation potential.

Copper-zinc superoxide dismutase (SOD-1) is an enzyme that is widely expressed in eukaryotic cells and performs a vital role in protecting cells against free radical damage. In mouse testis, three different sizes of SOD-1 mRNAs of about 0.73, 0.80, and 0.93 kilobases (kb) are detected. The 0.73-kb mRNA is found in early stages of male germ cells and in all somatic tissues. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase H digestions and Northern blot analyses reveal that the three SOD-1 mRNAs are derived from two transcripts, a ubiquitously expressed transcript and a post-meiotic transcript, which differ by 114-120 nucleotides. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5'-untranslated region. Using a probe derived from the 5'-untranslated region of the 0.93-kb SOD-1 mRNA, we have established that it originates from an alternative upstream promoter contiguous with the somatic SOD-1 promoter. Polysomal gradient analysis of the three mouse testis SOD-1 mRNAs reveals that the 0.93-kb SOD-1 mRNA is primarily non-polysomal, while the 0.80- and 0.73-kb SOD-1 mRNAs are mostly polysome associated. A faster migrating form of the 0.93-kb SOD-1 mRNA is present on polysomes as a result of partial deadenylation. In a cell-free translation system, the 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-1 mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-1 mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-1 mRNAs undergo adenylation changes, and one of the post-meiotic SOD-1 mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form.

Adenosine Monophosphate↗
PubMed1995

Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis.

RNA-binding proteins that bind to the 3' untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis.

Animals↗
PubMed1995

A mouse homologue of the Xenopus germ cell-specific ribonucleic acid/deoxyribonucleic acid-binding proteins p54/p56 interacts with the protamine 2 promoter.

Recent evidence indicates that a member of the Y box-binding family of transcriptional regulators is identical to p56, a predominant protein of messenger ribonucleoprotein complexes. The p56 protein is highly enriched in oocytes and testis, and a functional RNA binding mouse cytoplasmic homologue has been cloned and partially characterized. Because few potential testis-specific transcriptional regulators have been identified, the testis-enriched Y box-binding proteins represent trans-acting elements of a unique model system for the study of haploid gene expression. The 5' flanking region of the testis-specific, haploid-expressed mouse protamine 2 gene contains an element with a 9-of-12 nucleotide identity with the previously defined Y box consensus sequence. We have investigated the possible role of Y box-binding proteins in transcriptional regulation of protamine 2 using specific antibodies and DNA-protein binding assays. Western blot analyses with two different anti-p54/p56 antibodies demonstrate that a mouse homologue of Xenopus p54/p56 is present in transcriptionally active mouse testis nuclear extracts. Our results further indicate that the Xenopus Y box-binding proteins bind to an element 5' to the mouse protamine 2 gene. Similarly, binding of the mouse testis homologue to the protamine 2 Y box element is demonstrated by gel mobility shift and antibody supershift analyses. The demonstrated interactions between testis-enriched Y box-binding proteins and protamine 2 transcriptional control elements therefore represent a unique system for functional studies to determine the mechanism of regulation of haploid gene expression.

Animals↗
PubMed1995

Testis-brain RNA-binding protein, a testicular translational regulatory RNA-binding protein, is present in the brain and binds to the 3' untranslated regions of transported brain mRNAs.

Previous studies have demonstrated that a phosphoprotein in testis binds to transcript c, a sequence containing two highly conserved elements, Y and H, in the 3' untranslated region (UTR) of mouse protamine 2 mRNA (mP2) and represses its translation in vitro. When gel-retardation assays were performed with cytoplasmic extracts prepared from seven different mouse tissues, we found that brain in addition to testis contains a protein that binds to transcript c. Both the testis and brain proteins are found exclusively in the nonpolysomal fractions of their postmitochondrial extracts. The testis and brain proteins appear to be identical according to numerous criteria: the complexes they form with transcript c have identical mobility in native gels, identical optimal pH, identical lability to increased salt concentrations, identical chromatographic properties, identical molecular sizes as judged from UV crosslinking, and identical peptide mapping as revealed by V8 digestion of the UV crosslinked protein-RNA complexes. In addition to binding to the same conserved sequence in the 3'UTR of mP2, the phosphoprotein from testis and brain, hereafter called testis-brain RNA-binding protein (TB-RBP), also specifically binds to a similar sequence in the 3'UTR of brain Tau mRNA. Since TB-RBP binds to the 3'UTRs of several translationally regulated mRNAs in testis and since numerous transported brain mRNAs also contain the same conserved binding elements, we propose that TB-RBP plays a role in mRNA storage, translocation, and/or localization in brain and testis.

Ammonium Sulfate↗
PubMed1994

Heterogeneity in the 5' untranslated region of mouse cytochrome cT mRNAs leads to altered translational status of the mRNAs.

Previous studies have shown that the differential regulation of mouse somatic cytochrome c (cyt cS) and testicular cytochrome c (cyt cT) during spermatogenesis is accompanied by changes in mRNA length [Hake et al. (1990) Development, 110, 249-257]. When analyzed by polysomal gradient sedimentation, cytochrome cT sediments in two broad size classes: non-polysomal mRNAs are about 0.6 to 0.75 kb and polysomal mRNAs range from 0.7 to 0.9 kb. Both classes of mRNAs shorten to about 0.5 kb following deadenylation. Oligonucleotide-directed cleavage of the cytochrome cT RNAs by RNase H reveals that the size heterogeneity of cytochrome cT mRNAs resides in the 5' untranslated regions (UTRs). Ribonuclease protection assays reveal that multiple cytochrome cT mRNAs are transcribed from six different transcriptional start sites spanning a region of 59 nucleotides in the 5'UTR from +1 to +59. Transcripts derived from the first and second transcriptional initiation sites are not loaded onto polysomes as efficiently as those transcripts initiated from the other start sites. Each of the longer mRNAs has an upstream open reading frame, which starts at +8 and ends at +136 in the 5'UTR of the cytochrome cT transcript. Computer analysis suggests that the lengthened 5'UTR sequences allow additional hairpin structures to be formed. Since the upstream open reading frame and the additional stem loop structure are absent in the 5' UTRs of the cytochrome cT mRNAs initiated from the four downstream start sites, we suggest that these sequences in the two longest cytochrome cT transcripts hinder their loading onto polysomes.

Animals↗
PubMed1994

Synthesis and processing of mammalian protamines and transition proteins.

Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [3H]-arginine, [3H]-histidine, [35S]-cysteine, or [32P]-PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms.

Animals↗
PubMed1994

The genes encoding the somatic and testis-specific isotypes of the mouse cytochrome c genes map to paralogous regions of chromosomes 6 and 2.

Using mouse probes specific to cytochrome cs and to cytochrome cT, the single-copy genes encoding these two proteins have been mapped to paralogous chromosomal regions by analysis of restriction fragment length variants in interspecific crosses. The gene for cytochrome cs, Cycs, maps to a position between Tcrb and Cbl-1 on proximal mouse Chromosome 6, and the gene for cytochrome cT, Cyct, maps between Gad-1 and Sfpi-1 on mouse Chromosome 2.

Animals↗
PubMed1994

Poly(A)+ ribonucleic acids are enriched in spermatocyte nuclei but not in chromatoid bodies in the rat testis.

To determine whether male germ cells contain specific storage sites for poly(A)+ RNAs, in situ hybridizations were performed with sections of rat testis and a [3H]polyuridylic acid probe. The highest levels of poly(A)+ RNA were found in spermatocytes and round spermatids, while lower levels of poly(A)+ RNA were detected in spermatogonia, elongated spermatids, Sertoli cells, myoid cells, fibroblasts, macrophages, and Leydig cells. No poly(A)+ RNA was detected in residual bodies of elongated spermatids. At stages IX-XI of the seminiferous cycle, the nuclei and cytoplasm of pachytene spermatocytes contained approximately equal amounts of poly(A)+ RNA, suggesting nuclear RNA storage and/or a reduced processing rate of mRNA precursors at this stage of germ cell differentiation. To examine the distribution of poly(A)+ RNAs in subcellular components of testicular cells, electron microscope radioautography was used. In germ cells and Sertoli cells, poly(A)+ RNA was often seen free in the cytoplasm or associated with the endoplasmic reticulum and was only occasionally found associated with mitochondria, lysosomes, lipid inclusions, and axonemes. As previously reported for the mRNAs of transition protein 1 and protamine 1 [Morales et al., J Cell Sci 1991; 100:119-131], no compartmentalization of poly(A)+ RNAs was detected in the cytoplasm of round and elongated spermatids. No poly(A)+ RNA was detected in association with the radial body and in most sections, the chromatoid body did not contain any significant amounts of poly(A)+ RNA.

Animals↗
PubMed1993

Utilization of an alternative transcription initiation site of somatic cytochrome c in the mouse produces a testis-specific cytochrome c mRNA.

The differential regulation of somatic and testis-specific cytochromes c during spermatogenesis in the mouse is accompanied by changes in mRNA length (Hake, L. E., Alcivar, A. A., and Hecht, N. B. (1990) Development 110, 249-257). In spermatogenic stem cells through early meiotic cells, we detect four somatic cytochrome c (cyt cs) mRNAs of 1.3, 1.1, and 0.7-0.5 kilobases (kb), whereas in postmeiotic cells we detect a larger cyt cs mRNA of 1.7 kb. Oligonucleotide-directed RNase H cleavage of cyt cs mRNA revealed that the 1.7-kb mRNA contains over 1 kb of 5'-untranslated region which is not present in the four shorter cyt cs mRNAs. RNase protection assays indicate that this additional sequence arises from the utilization of an alternative transcription initiation site of the functional cyt cs gene which is 1085 base pairs upstream of the initiation site for the four shorter cyt cs mRNAs. To analyze the promoter for the 1.7-kb mRNA, a genomic clone containing the cyt cs gene and 5 kb of 5'-flanking DNA was isolated. Sequence comparison of the putative promoter region with promoters of other postmeiotically expressed genes reveals several conserved regions. Utilization of this alternative initiation site may be involved in the down-regulation of cytochrome cs during spermatogenesis.

Animals↗
PubMed1993

Cellular localization of the mRNAs of the somatic and testis-specific cytochromes c during spermatogenesis in the rat.

During mammalian spermatogenesis, two forms of cytochrome c, cytochromes cs and ct, are present in male germ cells. During meiosis, cytochrome ct begins to replace cytochrome cs. At least four size classes of cytochrome cs mRNA are expressed in all somatic cells and in early stages of male germ cells. In addition, a cytochrome cs transcript of 1.7 kB has been detected in rodent testes and is abundant in post meiotic male germ cells. Here we utilize "in situ" hybridization to define the cellular sites where the four ubiquitous cytochrome cs transcripts, the 1.7 kB cytochrome cs transcripts, and the testis-specific cytochrome ct transcripts are expressed in the rat. Low levels of cytochrome cs mRNAs are detected in Leydig cells, myoepithelial cells, Sertoli cells, all types of spermatogonia, and during meiotic prophase. The 1.7 kB cytochrome cs mRNA is first detected in late stages of meiotic prophase and reaches its highest levels in steps 1 to 9 spermatids. No cytochrome cs mRNAs are detected in spermatids between steps 10 to 19. Low levels of cytochrome ct mRNAs, initially detected in zygotene spermatocytes, reach maximal levels in round spermatids. For all three probes the majority of the silver grains are localized randomly throughout the cytoplasm, suggesting that neither the translating nor non-translating (the 1.7 kB mRNA) forms of cytochrome cs mRNA nor the cytochrome ct mRNAs are sequestered during spermatogenesis. The absence of cytochrome cs or ct mRNAs in steps 10-19 spermatids suggest that the cytochrome ct protein does not turn over rapidly in late stage male germ cells.

Animals↗
PubMed1993

Proteins homologous to the Xenopus germ cell-specific RNA-binding proteins p54/p56 are temporally expressed in mouse male germ cells.

Antibodies specific for the Xenopus oocyte cytoplasmic 6S mRNA-binding particle p54/p56 and antibodies against Xenopus germ cell DNA-binding protein FRG Y2 recognize two RNA-binding proteins of the mouse testis. The mouse testis proteins, estimated by SDS-PAGE to be about 48 and 52 kDa, form RNA-protein complexes with either translationally regulated or control RNAs, suggesting that they are sequence-independent RNA-binding proteins. The binding of the 48/52-kDa proteins to RNA is reduced by heparin. The expression of the 48/52-kDa mouse proteins is germ cell-specific and developmentally regulated in the testis with a maximal amount of the two proteins being detected in early postmeiotic cells (round spermatids), a cell type where many mRNAs are stored. The 48/52-kDa proteins are detected solely in the nonpolysomal fractions of postmitochondrial adult testis extracts and are not detected in extracts of brain, liver, or prepuberal testes from 12-day-old mice. We conclude that two RNA-binding proteins that appear to be immunological and functional homologues of the Xenopus germ cell-specific RNA/DNA-binding proteins p54/p56/FRG Y2 are present in male germ cells and form complexes with stored mRNAs.

Animals↗
PubMed1993

Tracing the incorporation of the sperm tail in the mouse zygote and early embryo using an anti-testicular alpha-tubulin antibody.

The mechanism of sperm tail incorporation and the fate of the tail during mouse fertilization and early embryogenesis were examined. Time-lapse video microscopy and anti-tubulin immunofluorescence show that the incorporation of the sperm tail, but not the sperm head, is sensitive to cytochalasin B (a microfilament inhibitor). Colcemid, a microtubule inhibitor, does not affect tail incorporation. High-resolution, low-voltage scanning electron microscopy demonstrates that the plasma membrane covering the sperm tail does not appear to fuse with the oocyte membrane during in vitro fertilization in the presence of cytochalasin. In control and colcemid-treated oocytes, the plasma membrane along the sperm tail, which is oriented tangential to the egg surfaces, appears to fuse with the oocyte membrane at multiple sites. An antibody to testicular alpha-tubulin detects sperm-derived, but not egg, microtubules and this has permitted us to trace the behavior and disappearance of the sperm tail during embryogenesis. Conventional and confocal microscopy show that following sperm incorporation, the tail often splays into multiple fibers. At the two-cell stage, the axoneme may be localized in either blastomere or it may be found to run through the midbody between both blastomeres. The tail appears to shorten by the 8-cell stage and is undetectable after the 16-32 cell stage. In morulae, tail fragments have been found in outer cells but not in inner ones, and fragments have not be found in blastocysts. These data suggest that microtubules of sperm and oocytes contain different isotypes of alpha-tubulin, nongenomic sperm-derived components survive at least to the morula stage of mouse development, and egg microfilaments are involved in the incorporation of the sperm tail but not the sperm head, which demonstrates that motility during sperm incorporation is different in mammals when compared to lower vertebrates and invertebrates.

Actin Cytoskeleton↗
PubMed1993

Binding of a phosphoprotein to the 3' untranslated region of the mouse protamine 2 mRNA temporally represses its translation.

The synthesis of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated during germ cell development by mRNA storage for about 7 days in the cytoplasm of differentiating spermatids. Two highly conserved sequences, the Y and H elements present in the 3' untranslated regions (UTRs) of all known mammalian protamine mRNAs, form RNA-protein complexes and specifically bind a protein of 18 kDa. Here, we show that translation of fusion mRNAs was markedly repressed in reticulocyte lysates supplemented with a mouse testis extract enriched for the 18-kDa protein when the mRNAs contained the 3' UTR of mouse protamine 2 (mP2) or the Y and H elements of mP2. No significant decrease was seen when the fusion mRNAs contained the 3' UTR of human growth hormone. The 18-kDa protein is developmentally regulated in male germ cells, requires phosphorylation for RNA binding, and is found in the ribonucleoprotein particle fractions of a testicular postmitochondrial supernatant. We propose that a phosphorylated 18-kDa protein plays a primary role in repressing translation of mP2 mRNA by interaction with the highly conserved Y and H elements. At a later stage of male gamete differentiation, the 18-kDa protein no longer binds to the mRNA, likely as a result of dephosphorylation, enabling the protamine mRNA to be translated.

Animals↗
PubMed1992

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.

Animals↗