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W F Marzluff

Publications and source records attributed to W F Marzluff.

At least 55 records · Page 3Linked to original sources
PubMed1992

The histone mRNA 3' end is required for localization of histone mRNA to polyribosomes.

The final step in mRNA biosynthesis is transport of the mRNA from the nucleus to the cytoplasm. Histone genes from which the 3' stem-loop has been deleted are transcribed to give RNAs with heterogeneous 3' ends. These RNAs are localized in the nucleus and are stable. Addition of the histone 3' processing signal either on short (< 250 nts) or long (> 1000 nts) transcripts restores 3' processing and transport of the mRNA to the cytoplasm. In addition chimeric histone-U1 snRNA genes which produced RNAs with either histone or U1 3' ends were analyzed. Transcripts which ended with U1 snRNA 3' ends were not efficiently localized to polyribosomes. However, transcripts containing the same sequences including the snRNA 3' end followed by the histone 3' end were present in the cytoplasm on polyribosomes. Taken together these results suggest that the histone 3' end is required for export of histone mRNA to the cytoplasm and association of the mRNA with polyribosomes.

Animals↗
PubMed1992

Two promoter elements are necessary and sufficient for expression of the sea urchin U1 snRNA gene.

The essential elements of the sea urchin L. variegatus U1 snRNA promoter were mapped by microinjection of a U1 maxigene into sea urchin zygotes. Two elements are required for expression: a distal sequence element (DSE) located between -318 and -300 and a proximal sequence element (PSE) centered at -55. Removal or alteration of other sequences conserved in different sea urchin snRNA U1 genes, including deletion of all sequence between -90 and -273, did not affect the expression. Sequences around the start site were not required for expression. Deletion of nucleotides between the PSE and the start site resulted in initiation inside the U1 coding region, suggesting that the PSE determines the start site of transcription. There is no obvious similarity between the sequences required for the sea urchin U1 snRNA expression and the sequences required for the expression of other sea urchin snRNAs.

Animals↗
PubMed1992

A conserved region in the sea urchin U1 snRNA promoter interacts with a developmentally regulated factor.

The expression of the sea urchin L. variegatus U1 snRNA gene is temporally regulated during embryogenesis. Using a microinjection assay we show that a region between 203 and 345 nts 5' of the gene is required for expression. There are four conserved regions between two sea urchin species in the 345 nts 5' to the U1 gene. One region, located at about -300, binds a protein factor which is present in blastula but not gastrula nuclei. Three other potential protein binding sites within the first 200 nts 5' to the gene have been identified using a mobility shift assay and/or DNase I footprinting. Two of these regions bind factors which are not developmentally regulated and one binds a factor which is developmentally regulated. It is likely that the factor which binds at -300 is involved in expression and developmental regulation of the sea urchin U1 snRNA gene.

Animals↗
PubMed1992

Molecular modeling to predict the structural and biological effects of mutations in a highly conserved histone mRNA loop sequence.

The 3'-end of histone mRNAs contains a highly conserved sequence motif which is believed to form a 6 base pair stem and a 4 base loop. These sequences are involved in both the efficiency of 3'-end formation and stability of the mature histone mRNA. We have modeled four stem basepairs and the loop portion of this structure using the wildtype sequences and several mutant sequences. A structure for the wildtype stem-loop is proposed that is based on energy minimization using a representative wildtype sequence and comparison with structures obtained using naturally occurring mutations which do not alter loop function. A wildtype structure is proposed in which the top basepair of the stem is broken, forming a six base loop. Mutant sequences with altered bases in the loop and in the stem were also modeled. The effect of these mutations on the proposed wildtype structure is discussed and possible biological consequences considered.

Base Sequence↗
PubMed1992

Characterization of two developmentally regulated sea urchin U2 small nuclear RNA promoters: a common required TATA sequence and independent proximal and distal elements.

The promoters of two U2 small nuclear RNA genes isolated from the sea urchin Lytechinus variegatus were mapped by microinjection of genes into sea urchin zygotes. One gene, LvU2E, is expressed only in oocytes and embryos and is found in a tandemly repeated gene set, while the other gene, LvU2L, is a single-copy gene and is expressed in embryos and somatic cells. The promoters each contain a TATA sequence at -25 which is required for expression, a proximal sequence element (PSE) centered at -55 required for expression, a sequence at -100 which couples the core promoter (PSE plus TATA box) to the upstream element, and an upstream sequence which stimulates expression fourfold. The PSE together with the TATA sequence is sufficient to determine the transcription start site. There is no sequence similarity between the -100 and PSE sequences of the two genes. The -100 sequences can be interchanged between the two genes. The LvU2E PSE functions in the context of the LvU2L gene, but the LvU2L PSE functions poorly in the context of the LvU2E gene.

Animals↗
PubMed1992

Formation of the 3' end of sea urchin U1 small nuclear RNA occurs independently of the conserved 3' box and on transcripts initiated from a histone promoter.

The formation of the 3' end of vertebrate small nuclear RNAs (snRNAs) requires that transcription initiate from an snRNA promoter. There is a loosely conserved required box 5 to 20 nucleotides (nt) 3' of the gene. The sea urchin snRNA genes contain promoter elements different from those of the vertebrate snRNAs. They also contain a characteristic 3' 15-nt sequence which is conserved among different sea urchin snRNA genes. We used microinjection of sea urchin U1 snRNA genes into sea urchin zygotes to define the sequence requirements for U1 snRNA 3'-end formation. Surprisingly, the conserved 3' box is not required for efficient 3'-end formation in vivo. Deletion analysis reveals that the 6 nt immediately 3' of the U1 snRNA are involved in 3'-end formation. Substitution analysis revealed that either these 6 nt 3' of the U1 RNA or the conserved 3' box could direct 3'-end formation. Transcripts initiated from a histone H4 promoter formed U1 3' ends about 50% as efficiently as transcripts initiated from the U1 promoter, even when the U1 end was placed in tandem with a histone 3'-processing signal, suggesting that transcription from an snRNA promoter is not necessary for formation of the 3' end of sea urchin U1 snRNA.

Animals↗
PubMed1991

Different complexes are formed on the 3' end of histone mRNA with nuclear and polyribosomal proteins.

Specific protein-RNA complexes are formed by incubating a synthetic histone mRNA 3' end (a 30 nucleotide stem-loop structure) RNA with extracts of either nuclei or polyribosomes. The complex formed between the stem-loop and nuclear proteins has a lower electrophoretic mobility than the complex formed between the stem-loop and polyribosomal proteins. Binding of the synthetic 3' end by both polyribosomal and nuclear proteins is abolished when two of the conserved uridine residues in the loop are replaced with adenosines. UV crosslinking of the protein complexes to the synthetic RNA resulted in transferring radiolabel to similar sized proteins, 50 kD, in both the nuclear and polyribosomal extracts.

Animals↗
PubMed1991

The U1 snRNA gene repeat from the sea urchin (Strongylocentrotus purpuratus): the 70 kilobase tandem repeat ends directly 3' to a U1 gene.

Lambda phage clones containing multiple copies of the 1.1 kb tandemly repeated unit of the sea urchin (S. purpuratus) U1 RNA genes were isolated from a gene library. The 1.1 kb repeat unit encodes a single copy of the predominant U1 RNA expressed in oocytes and embryos prior to the blastula stage. The tandem repeat unit is about 80 kb in size and is probably present one time per haploid genome as judged by pulsed-field electrophoresis of sperm DNA digested with restriction enzymes which do not cut in the repeat unit. Two of the phage contained DNA flanking the repeat unit as well as several repeat units. The tandem repeat unit ends just 3' to the U1 coding region. There is only limited homology in the 5' flanking region with U1 snRNA genes from the sea urchin L. variegatus.

Animals↗
PubMed1991

Isolation and characterization of developmentally regulated sea urchin U2 snRNA genes.

Genes encoding the U2 snRNA have been isolated from the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. Representatives of tandemly repeated gene sets have been isolated from both sea urchin species and a unique U2 gene has also been isolated from L. variegatus. The sequence of the U2 snRNA encoded by the tandemly repeated genes differs in two nucleotides between S. purpuratus and L. variegatus. The unique U2 gene from L. variegatus encodes the same U2 RNA as the tandemly repeated genes. There is a change in the U2 genes expressed between morula and pluteus embryos as judged by a change in the U2 RNA sequence in S. purpuratus embryos. The tandemly repeated genes were expressed at a higher rate in blastula than in gastrula stage relative to the single-copy gene, when the two genes were injected into sea urchin zygotes.

Animals↗
PubMed1991

An intact histone 3'-processing site is required for transcription termination in a mouse histone H2a gene.

A transcription termination site has been characterized between the mouse histone H2a-614 and H3-614 genes. There is a poly(A)- RNA present in small amounts in the nucleus which ends 600 nucleotides 3' to the H2a-614 gene. Nuclear transcription studies demonstrate that transcription extends at least 600 nucleotides 3' to the gene but is greatly reduced 700 nucleotides 3' to the gene. If all or part of the normal 3'-processing signal, consisting of the stem-loop and the U7 small nuclear ribonucleoprotein binding site, is deleted, transcription then continues past the putative termination site and RNAs which end at the 3' end of the downstream H3-614 gene accumulate. Insertion of a 150-nucleotide fragment containing the termination site between the histone 3' end and downstream polyadenylation sites reduces usage of polyadenylation sites 85 to 90%. Taken together these results suggest there is a transcription termination site which requires an intact histone 3'-processing signal to function.

Animals↗
PubMed1991

Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps.

The levels of histone mRNA increase 35-fold as selectively detached mitotic CHO cells progress from mitosis through G1 and into S phase. Using an exogenous gene with a histone 3' end which is not sensitive to transcriptional or half-life regulation, we show that 3' processing is regulated as cells progress from G1 to S phase. The half-life of histone mRNA is similar in G1- and S-phase cells, as measured after inhibition of transcription by actinomycin D (dactinomycin) or indirectly after stabilization by the protein synthesis inhibitor cycloheximide. Taken together, these results suggest that the change in histone mRNA levels between G1- and S-phase cells must be due to an increase in the rate of biosynthesis, a combination of changes in transcription rate and processing efficiency. In G2 phase, there is a rapid 35-fold decrease in the histone mRNA concentration which our results suggest is due primarily to an altered stability of histone mRNA. These results are consistent with a model for cell cycle regulation of histone mRNA levels in which the effects on both RNA 3' processing and transcription, rather than alterations in mRNA stability, are the major mechanisms by which low histone mRNA levels are maintained during G1.

Animals↗
PubMed1991

A common transcriptional activator is located in the coding region of two replication-dependent mouse histone genes.

There is a region in the mouse histone H3 gene protein-encoding sequence required for high expression. The 110-nucleotide coding region activating sequence (CRAS) from codons 58 to 93 of the H3.2 gene restored expression when placed 520 nucleotides 5' of the start of transcription in the correct orientation. Since identical mRNA molecules are produced by transcription of the original deletion gene and the deletion gene with the CRAS at -520, effects of the deletions on mRNA stability or other posttranscriptional events are completely ruled out. Inversion of the CRAS sequence in its proper position in the H3 gene resulted in only a threefold increase in expression, and placing the CRAS sequence 5' of the deleted gene in the wrong orientation had no effect on expression. In-frame deletions in the coding region of an H2a.2 gene led to identification of a 105-nucleotide sequence in the coding region between amino acids 50 and 85 necessary for high expression of the gene. Additionally, insertion of the H3 CRAS into the deleted region of the H2a.2 gene restored expression of the H2a gene. Thus, the CRAS element has an orientation-dependent, position-independent effect. Gel mobility shift competition studies indicate that the same proteins interact with both the H3 and H2a CRAS elements, suggesting that a common factor is involved in expression of histone genes.

Animals↗
PubMed1991

Expression of histone-U1 snRNA chimeric genes: U1 promoters are compatible with histone 3' end formation.

Chimeric genes which fuse the mouse histone H2a gene and the mouse U1b gene were constructed and introduced into CHO cells by cotransfection. In the UH genes, the U1b gene promoter and the start of the U1b gene were fused to the H2a gene in the 5' untranslated region. In the HU genes, the U1b 3' end was inserted into the 3' untranslated region of the H2a gene replacing the normal histone 3' end. Transcripts from the UH genes initiated at the start of the U1 gene and ended at the normal histone 3' end. Transcripts from the HU chimeric genes did not end at the U1 3' end but extended at least 80 nucleotides further and had heterogeneous 3' ends. Placing both a U1 snRNA promoter and a U1 snRNA 3' end around a histone coding region resulted in transcripts which initiate and terminate at the appropriate U1 ends. These results are consistent with previous reports that formation of the U1 3' ends require U1 promoters, but indicate that the histone 3' end can be formed on transcripts initiating at U1 promoters. The transcripts initiated at the U1 start site and ending at the histone 3' end are present on polyribosomes and show proper posttranscriptional regulation.

Animals↗
PubMed1990

Introns in histone genes alter the distribution of 3' ends.

Chimeric genes were constructed which contained either a histone or globin promoter, a human alpha-globin coding region as a cDNA or containing one or both intervening sequences, and the 3' end of a mouse histone H2a gene. The genes were introduced into mouse L cells or Chinese Hamster Ovary cells. The genes containing at least one intervening sequence produced two mRNAs in about equal amounts, one which ended at a cryptic polyadenylation site 33 nucleotides 3' to the normal histone mRNA 3' end and one which ended at the normal histone 3' end. In contrast, the same construct containing a globin cDNA yielded mRNA ending only at the correct histone 3' end. Similar proportions of polyadenylated and non-polyadenylated mRNA were obtained when the cryptic polyadenylation signal was replaced with the globin polyadenylation signal. More than 90% of the transcripts were accurately spliced. All of the unspliced transcripts had histone 3' ends.

Animals↗
PubMed1990

U3 snRNPs and nucleolar development during oocyte maturation, fertilization and early embryogenesis in the mouse: U3 snRNA and snRNPs are not regulated coordinate with other snRNAs and snRNPs.

U3 small nuclear ribonucleic acids (snRNA) and U3 small nuclear ribonucleoprotein (snRNP), which are thought to be responsible for ribosomal RNA processing, are quantitated and localized during oocyte maturation, fertilization, and early embryogenesis in the mouse. On the basis of Northern blot and nuclease protection experiments, it is estimated that there are about 5 x 10(4) U3 snRNA molecules in an ovulated oocyte and in a two-cell embryo. This number then increases roughly 50-fold to 2.7 x 10(6) molecules per embryo by the blastocyst stage. At all stages of development U3 snRNP antigens colocalize with nucleoli, as defined by differential interference contrast microscopy and an antibody to a nucleolar epitope. The synthesis and distribution of U3 snRNA and U3 snRNP follow a pattern independent from other major U snRNPs and snRNAs.

Animals↗