William F. Marzluff

The stem-loop structure at the 3' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability.

Chimeric genes were made by fusing mouse histone genes with a human alpha-globin gene. The genes were introduced into mouse L cells and the stability of the chimeric mRNAs was measured when DNA synthesis was inhibited. An mRNA containing all the globin coding sequences and the last 30 nucleotides of the histone mRNA was degraded at the same rate as histone mRNA.

Multiple regulatory steps control histone mRNA concentrations

Abstract The abundance of histone mRNAs in mammalian cells varies with the cell cycle. Both transcriptional and post-transcriptional regulatory mechanisms are involved, with mRNA degradation playing an important role.

Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation.

Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both Us to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.

Differential expression of two clusters of mouse histone genes

The mouse histone mRNAs coded for by three different cloned DNA fragments have been characterized. Two of these cloned DNA fragments, MM221 and MM291, located on chromosome 13, code for H3, H2b and H2a histone mRNAs, which are expressed at low levels in cultured mouse cells and fetal mice. The other DNA fragment, MM614, located on chromosome 3, codes for an H3 and an H2a mRNA, which are expressed at high levels in these cells. The mRNAs for each histone protein share common coding region sequences, while the untranslated regions of all the genes have diverged significantly, as judged by S1 nuclease mapping. Amino acid substitutions in some H3, H2a and H2b proteins are detected as internal cleavages in the S1 nuclease maps. All of these genes code for replication variant histone mRNAs, which are regulated in parallel with DNA synthesis.

Rapid reversible changes in the rate of histone gene transcription and histone mRNA levels in mouse myeloma cells

The levels of histone mRNAs are reduced 90 to 95% after treatment of mouse myeloma cells with inhibitors of DNA synthesis which disrupt deoxynucleotide metabolism. In contrast, novobiocin, which inhibits DNA synthesis but does not alter deoxynucleotide metabolism, did not alter histone mRNA levels. Upon reversing the inhibition by fluorodeoxyuridine by feeding with thymidine, histone mRNA levels are restored to control levels within 40 to 60 min. The rate of histone gene transcription is reduced 75 to 80% within 10 min after treatment with fluorodeoxyuridine and increased to control levels within 10 min after refeeding with thymidine. Inhibition of protein synthesis with cycloheximide or puromycin in cells which had been treated with fluorodeoxyuridine resulted in an increase of histone mRNA levels. This was partly due to an increase in the rate of transcription. The data indicate that both transcription and mRNA degradation are linked to deoxynucleotide metabolism. Continued protein synthesis is necessary for maintaining the inhibition of histone gene transcription.

HMG-proteins 1 and 2 are required for transcription of chromatin by endogenous RNA polymerase.

Summary Chromatin prepared from isolated mouse myeloma cell nuclei faithfully transcribes RNA using endogenous RNA polymerase. Extraction of the chromatin with 0.35M KC1 greatly reduces the ability of the chromatin to transcribe RNA, but RNA synthesis is restored by adding back the extracted material. The major proteins extracted under these conditions are the high mobility group proteins — HMG 1 and 2. When purified HMG proteins 1 and 2 were added back to the extracted chromatin preparations total RNA synthesis was stimulated 3 to 5 fold. The synthesis of the RNA polymerase III products, 5S rRNA and tRNA precursors, was also stimulated the same amount.

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.

Sequences of four mouse histone H3 genes: implications for evolution of mouse histone genes.

SummaryThe sequences of four histone H3 genes coding for the replication variant proteins H3.1 and H3.2 have been determined. Three of these genes, two coding for H3.1 proteins and one for an H3.2 protein, are located on chromosome 13 and expressed at low levels. The fourth gene, encoding an H3.2 protein, is located on chromosome 3 and expressed at a high level. The coding regions of the three genes on chromosome 13 are more similar to each other than to the H3 gene on chromosome 3, and equally divergent from it, suggesting that either gene duplication or gene conversion has occurred since the genes were dispersed onto two chromosomes. A 14-base sequence including the CCAAT sequence and located 5′ to the genes on chromosome 13 has been conserved. The histone H3 gene on chromosome 3 has multiple potential binding sites for the Sp1 transcription factor. The coding regions show >95% conservation among the four genes. This is due to the strict pattern of codon usage and the presence of two long (>60 base) regions of completely conserved nucleic acid sequence. These conserved regions in the coding sequence may have an important functional role at the mRNA or DNA level.

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%.

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1(0) protein was predominant in G0 cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.