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W F Marzluff

Publications and source records attributed to W F Marzluff.

At least 73 records · Page 4Linked to original sources
PubMed1989

A developmental switch in sea urchin U1 RNA.

The sequence of U1 RNA has been determined in the eggs and embryos of two sea urchins, Lytechinus variegatus and Strongylocentrotus purpuratus. In both species the sequence of the U1 RNA changes as the embryos progress through development. The sequence of the major U1 RNA in the eggs of the two species differs in two nucleotides, while the sequence of the U1 RNA present in the late embryos and somatic tissue is identical in the two species. The U1 RNA in eggs and early embryos is primarily derived from the tandemly repeated gene set, which is not expressed in somatic tissues.

Animals↗
PubMed1989

The early and late sea urchin histone H4 mRNAs respond differently to inhibitors of DNA synthesis.

The effect of inhibiting DNA synthesis on the concentration of the alpha-histone mRNA and the late histone mRNAs in sea urchin embryos was measured. The alpha-histone mRNA concentrations did not change, while the late histone mRNA concentrations were rapidly reduced at the three developmental stages (morula, blastula, and mesenchyme blastula) tested. The rapid degradation of the late histone mRNAs was prevented when protein synthesis was inhibited.

Animals↗
PubMed1989

A region in the coding sequence is required for high-level expression of murine histone H3 gene.

Replication-dependent histone genes are expressed at high rates in S phase to provide the histone proteins required for chromosomal replication. Two genes, an H2a and H3 gene, located on chromosome 3 in the mouse and cloned together in a single 3-kilobase (plasmid MM614) restriction fragment are highly expressed. By transfecting mouse histone gene constructs into Chinese hamster ovary cells, we have identified a 110-nucleotide region within the coding sequence of the H3.2-614 gene that is required for high-level expression. Deletion of this region reduces expression of the gene by 20-fold. Additionally, the histone-coding region activates the human alpha-globin promoter, which is normally not expressed well in Chinese hamster ovary cells. Similar results with deletion constructions involving the H2a-614 gene suggest that an intragenic region plays an important role in transcription of these genes.

Animals↗
PubMed1989

Expression of the U1 RNA gene repeat during early sea urchin development: evidence for a switch in U1 RNA genes during development.

The majority of the genes for U1 RNA are organized in tandemly repeated units in the sea urchin. To assess the level of expression of these genes in the sea urchin Lytechinus variegatus, we measured the transcription of sequences 3' to the gene. The tandemly repeated U1 genes are expressed in morula and continue to be expressed at high rates until 2 hr after hatching, at which time the rate of expression of all the U1 genes and the tandemly repeated U1 genes declines sharply. By the gastrula stage the synthesis of total U1 RNA has declined by a factor of 8. The major tandemly repeated genes are inactive by this time, although other U1 genes remain active. The sequence of U1 RNA synthesized late in embryonic development differs from the sequence of U1 RNA encoded by the tandemly repeated set of U1 RNA genes, indicating that there must be other U1 RNA genes that are active late in embryonic development.

Animals↗
PubMed1989

The efficiency of 3'-end formation contributes to the relative levels of different histone mRNAs.

Sequences at both the 5' and 3' ends of mouse histone genes contribute to the expression of individual genes. The 3' sequences required for high expression of the mouse H2a-614 gene are the same as the sequences required for 3'-end formation. When these sequences were substituted for the 3' end of the poorly expressed H2a-291 gene, expression of the H2a-291 gene was increased fivefold. A 65-nucleotide fragment containing the H2a-614 3' processing signal increased expression of the H2a-291 gene when it was placed in the proper orientation downstream of the H2a-291 3' end. The only mRNAs that accumulated from this gene ended at the H2a-291 3' end, which suggests that the transcript is sequentially processed. In an in vitro processing system, the different histone 3' ends showed different processing efficiencies, which correlated with their expression in cells. These results suggest that the efficiency of processing is important in determining the steady-state levels of individual mouse histone mRNAs.

Animals↗
PubMed1989

The small nuclear RNAs for pre-mRNA splicing are coordinately regulated during oocyte maturation and early embryogenesis in the mouse.

The abundance and localization of snRNAs and snRNPs involved in processing and splicing of pre-mRNA has been studied during early mouse embryogenesis. The amount of U1, U2, U4, U5 and U6 RNA remains relatively constant between the postovulatory oocyte and 2-cell stage but increases three- to ten-fold in quantity between the 2-cell and blastocyst stages. Localization was examined by in situ hybridization with U1, U2 and U6 riboprobes and immunofluorescence microscopy using a monoclonal antibody to snRNP antigens. The snRNAs and snRNPs are primarily localized to the germinal vesicle in the preovulatory oocyte but are released and diluted within the cytoplasm of the oocyte during germinal vesicle breakdown and meiotic maturation. They subsequently relocalize to both pronuclei following fertilization and the nuclei of the 2-cell embryo following the first cleavage division. Since the amount of snRNA is constant during the first cleavage, the small amount of pre-mRNA that is synthesized at the time of transcriptional activation in the 2-cell embryo may be spliced and processed by snRNPs of maternal origin.

Animals↗
PubMed1988

Structure of an unusual sea urchin U1 RNA gene cluster.

Genomic clones containing multiple copies of the Lytechinus variegatus U1 gene have been isolated from a gene library in the phage lambda EMBL3. These clones contain both types of U1 RNA gene repeats interspersed in the same 15-kb fragment. In addition, about 1/3 of the repeat units contain a 260-bp insert 460 bp prior to the first nucleotide of the U1 RNA sequence. The inserted sequence is abundant in the sea urchin genome as judged by Southern blots of genomic DNA. There are no repeated sequences flanking the insert. The insert occurs at the same position in the highly conserved 5'-flanking region at which a deletion has previously been reported.

Animals↗
PubMed1988

Mapping and gene order of U1 small nuclear RNA, endogenous viral env sequence, amylase, and alcohol dehydrogenase-3 on mouse chromosome 3.

Linkage was established between a number of genes that map on chromosome 3 by studying the distribution patterns of DNA polymorphisms and protein electrophoretic mobility polymorphisms in recombinant inbred (RI) strains of mice. This analysis resulted in the following suggested gene order between the newly assigned genes and previously mapped genes: gamma-fibrinogen (Fgg), Xmmv-22 of mink cell focus-inducing (MCF) virus, U1b small nuclear RNA gene cluster (Rnu-1b), amylase (Amy-1,2), cadmium resistance (cdm), alcohol dehydrogenase-3 (Adh-3), alcohol dehydrogenase-1 (Adh-1). In situ hybridization to chromosome spreads confirmed the assignment of the Ulb small nuclear RNA (snRNA) gene cluster and the gamma-fibrinogen gene to the center of chromosome 3.

Alcohol Dehydrogenase↗
PubMed1988

Localization and expression of U1 RNA in early mouse embryo development.

We have studied the accumulation and localization of U1 RNA during mouse embryo development by in situ hybridization with a U1 RNA probe and immunofluorescence microscopy using a mouse monoclonal antibody to U1 snRNP. There is a substantial amount of U1 RNA present in the oocyte that is present in both the germinal vesicle and the cytoplasm although the concentration is higher in the nuclear compartment. Following the germinal vesicle breakdown that accompanies ovulation and meiotic maturation, the U1 RNA is uniformly distributed throughout the unfertilized oocyte. In the fertilized egg, the silver grain density from in situ hybridization is higher over pronuclei and this enrichment is maintained at the two-cell and later stages. Similar results were obtained for the distribution of the U1 snRNP as assayed by immunofluorescence microscopy: U1 RNA is predominantly localized in all nuclei except polar body nuclei. The U1 RNA in the oocyte and two-cell embryo is predominantly (greater than 85%) U1a RNA. By the eight-cell stage there is a two to three-fold increase in the amount of total U1 RNA and the proportion of U1b RNA has increased to about 40%. The amount of U1 RNA continues to increase through the blastocyst stage and the proportion of the U1b RNA increases to 60%.

Animals↗
PubMed1988

Differential expression of individual members of the histone multigene family due to sequences in the 5' and 3' regions of the genes.

Histone proteins are encoded by a multigene family. The H3.2(614) and H2a(614) genes are present as single copies which are expressed at high levels, accounting for 30 to 40% of the H3 and H2a mRNAs, respectively, in different types of mouse cells. The other genes which have been isolated each contribute only a very small amount to the total type-specific mRNA pool. We demonstrate here that the differences in the level of expression of these genes are partly due to differences in their transcription rates. To investigate the sequences responsible for these differences in expression among the members of each family, we carried out DNA-mediated gene transfer experiments with both intact and chimeric histone genes. The 5' region of a highly expressed gene [H3.2(614) or H2a(614)] was attached to the 3' region of a histone gene which was expressed at low levels (H3-221 or H2a-291) and vice versa. The results show that sequences in both the 5' and 3' regions of the H3.2(614) and H2a(614) genes contribute to their high level of mRNA production by two independent mechanisms. The effect of the 3' sequences on mRNA accumulation has been narrowed to a 65-base-pair region including the 3'-terminal palindrome and downstream signal implicated in mRNA processing.

Animals↗
PubMed1987

Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region.

Animals↗
PubMed1987

Translation is required for regulation of histone mRNA degradation.

When DNA synthesis is inhibited, the mRNAs coding for the replication-dependent histone proteins are selectively destabilized. The histone genes have been altered and reintroduced into tk- mouse L cells by cotransfection with the herpesvirus thymidine kinase gene. Two features of the mRNA are necessary for regulation of degradation: first, the hairpin loop must be present at the 3' end of the histone mRNA; and second, the histone mRNA must be capable of being translated to within 300 nucleotides of the 3' end of the RNA. Polyadenylated histone mRNAs are stable, as are histone mRNAs that contain in-frame termination codons early in the coding region or 500 nucleotide 3' untranslated regions with a normal hairpin loop at the 3' end.

Amino Acid Sequence↗
PubMed1987

Characterization of mouse H3.3-like histone genes.

We designed a strategy to select genomic clones of mouse replication-independent H3.3 histone genes. We obtained three clones which met our selection criteria for being H3.3 genes. Upon sequencing two of these clones we found that they were unlike previously isolated chicken H3.3 clones: they code for several unpredicted amino acid substitutions and contain no introns in the coding regions. We showed by S1 nuclease assays that these genes are protected by mRNAs that have expression characteristics of H3.3 mRNA. The protection data and nucleotide sequence analysis show that the H3.3 transcripts can be processed at one of four cleavage/polyadenylation sites. We show that these genes probably evolved through reverse transcription intermediates, and are processed pseudogenes which are no longer under selective pressure. The 5' and 3' transcribed, nontranslated sequences show extensive homology to those of a human cDNA clone, and we suggest that these sequences may be required for appropriate regulation of expression of H3.3 genes.

Animals↗
PubMed1987

Coupling of replication type histone mRNA levels to DNA synthesis requires the stem-loop sequence at the 3' end of the mRNA.

The role of the 3' end of mRNA in coupling between the level of histone mRNAs and DNA synthesis was examined. We introduced modified mouse histone H3 genes into mouse fibroblasts and studied the regulation of several different H3 mRNAs that are not terminated with a normal histone 3' end. In two cases, the stem-loop sequences were deleted from the mRNAs and replaced either by 3' sequences flanking the H3 gene or by globin 3' untranslated region sequences including the polyadenylylation signal. In the former case, approximately equal to 50% of the modified mRNA was polyadenylylated, whereas in the latter case all of the mRNA had a polyadenylylated terminus. In contrast to the normal histone mRNAs, these mRNAs, including the nonadenylylated form, were stable when DNA synthesis was inhibited with several drugs. The levels of two other histone mRNAs, each containing the stem-loop sequences as an internal part of the mRNA, also were stable when DNA synthesis was inhibited. These results indicate that the posttranscriptional coupling of histone mRNA levels to DNA synthesis requires the presence of the stem-loop sequences at the 3' end of the mRNA.

Animals↗
PubMed1987

Sea urchin maternal and embryonic U1 RNAs are spatially segregated in early embryos.

We have used in situ hybridization and cell fractionation methods to follow the distribution of U1 RNA and immunofluorescence microscopy to follow the distribution of snRNP proteins in oocytes, eggs, and embryos of several sea urchin species. U1 RNA and U1-specific snRNP antigens are concentrated in germinal vesicles of oocytes. Both appear to relocate after oocyte maturation because they are found primarily, if not exclusively, in the cytoplasm of mature unfertilized eggs. This cytoplasmic residence is maintained during early cleavage and U1 RNA is first detectable in nuclei of micromeres at the 16-cell stage. Between morula and gastrula stages the steady-state concentrations of both RNA and antigens gradually increase in nuclei and decrease in cytoplasm. Surprisingly, analysis of the distribution of newly synthesized U1 RNA shows that it does not equilibrate with the maternal pool. Instead new transcripts are confined to nuclei, while cytoplasmic U1 RNAs are of maternal origin. This lack of equilibration and the conversion of maternal U1 RNAs from nuclear species in oocytes to cytoplasmic in embryos suggests that these RNPs (or RNAs) are structurally altered when released to the cytoplasm at oocyte maturation.

Animals↗
PubMed1987

Synthesis of U1 RNA in isolated mouse cell nuclei: initiation and 3'-end formation.

The transcription of U1 RNA genes was studied in isolated nuclei from mouse myeloma cells. Using a cloned U1b gene as a probe, we showed that isolated nuclei synthesize both U1b and U1a RNA. The U1 RNAs were initiated in vitro, as measured by incorporation of adenosine 5'-O-(2-thiotriphosphate) into U1 RNA. There was transcription of the 3'-flanking region but no transcription of regions directly 5' to the U1 genes. In addition to U1 RNAs of the correct length which were released from the nuclei, there were larger RNAs, presumably resulting from transcription into the 3'-flanking region, which were retained in the nuclei. Chase experiments showed that these longer transcripts were not precursors to mature U1 RNA, a finding consistent with the idea that 3'-end formation is coincident with transcription. During the chase, there was maturation of the 3' ends of U1a and U1b RNAs from slightly longer precursors. In addition to accurate transcription of U1 RNA, there was also synthesis of U2 and U3 RNA. All three of these RNAs were transcribed by RNA polymerase II, as measured by their sensitivity to alpha-amanitin.

Adenosine Triphosphate↗