Search

We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants. Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198. Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes. These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure. Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD. Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD.

Histone RNA 3′ end formation occurs through a specific cleavage reaction that requires, among other things, base‐pairing interactions between a conserved spacer element in the pre‐mRNA and the minor U7 snRNA present as U7 snRNP. An oligonucleotide complementary to the first 16 nucleotides of U7 RNA can be used to characterize U7 snRNPs from nuclear extracts by native gel electrophoresis. Using similar native gel techniques, we present direct biochemical evidence for a stable association between histone pre‐mRNA and U7 snRNPs. Other complexes formed in the nuclear extract are dependent on the 5′ cap structure and on the conserved hairpin element of histone pre‐mRNA, respectively. However, in contrast to the U7‐specific complex, their formation is not required for processing. Comparison of several authentic and mutant histone pre‐mRNAs with different spacer sequences demonstrates that the formation and stability of the U7‐specific complex closely follows the predicted stability of the potential RNA‐RNA hybrid. However, this does not exclude a stabilization of the complex by U7 snRNP structural proteins.

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.

In transient expression studies after DNA transfection of HeLa cells, the mouse U7 gene produces only approximately 30% of the RNA produced by a mouse U1b gene. This difference persists even when the transfected genes have all their 5′ and 3′ flanking sequences exchanged suggesting a post‐transcriptional effect. When the special U7 Sm binding site is mutated to a consensus derived from the major snRNAs (Sm‐opt), the U7 RNA level increases 4‐ to 5‐fold, whereas no RNA is detected from a U7 gene with a non‐functional Sm binding site (Sm‐mut). Moreover, U1b genes with the U7 Sm binding site yield reduced RNA levels. The Sm‐opt site also alters the cellular behaviour of the corresponding U7 snRNA. It accumulates to a higher level in the nucleus than wild type U7 RNA, and is better immunoprecipitable with anti‐Sm antibodies. Injection experiments in Xenopus oocytes indicate that the U7 genes with either Sm‐opt or Sm‐mut sites produce similar amounts of RNA as wild type U7, but that they differ in opposing ways in the processing of precursors to mature size U7 snRNA and in nuclear accumulation. However, in reconstitution experiments using Xenopus oocytes, we show that U7 Sm‐opt RNA, despite its efficient nuclear accumulation, is not active in 3′ processing of histone pre‐mRNA, whereas wild type U7 RNA is assembled into functional snRNPs, which correctly process histone pre‐mRNA substrate. This suggests a functional importance of the special U7 Sm sequence.

In vitro-synthesized transcripts of the sea urchin histone H2A gene with 3' extensions are efficiently and rapidly processed to H2A mRNA with faithful 3' ends in Xenopus laevis oocyte nuclei. Processing requires the presence of a histone-specific dyad symmetry element and of H2A-proximal spacer sequences in the precursor RNA. In DNA injection experiments with a processing-deficient H2A mutant, the transcription products appear to terminate heterogeneously in the first 100-200 base pairs of the post-H2A spacer. Processing of synthetic H3 RNA precursors requires the prior injection of a 60-nucleotide RNA from sea urchin embryos that seems to be a component of a small nuclear ribonucleoprotein.

Fragments from the 3′ end of a mouse histone H4 gene, when introduced into transcription units controlled by the SV40 early promoter, yield correctly processed RNA with histone‐specific 3′ ends, both in monkey and mouse cell lines. The processed RNA is regulated in parallel with endogenous H4 mRNAs in 21‐Tb cells, a temperature‐sensitive mouse mastocytoma cell cycle mutant that is specifically blocked in G1 phase at the non‐permissive temperature. Mutational analyses of the H4 gene fragment indicate that the minimal sequences for this regulation and for RNA 3′ processing are both contained within the same 80 bp. This fragment contains two histone‐specific, highly conserved sequence elements that are located at the 3′ end of histone mRNA and in the adjacent spacer region, respectively. Our data suggest that the observed cell cycle regulation is achieved either at RNA 3′ processing or at some later step involving the conserved 3′‐terminal sequence element of mature histone mRNA.

In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.

We have utilized a recombinant vector system designed to study gene control elements by fusing them to the Escherichia coli galactokinase gene (galK) to examine the effects of gene expression that result from introducing changes into the 5' noncoding leader region of the galK transcription unit. We varied the length of the 5' leader region and found no effect on galK expression, provided that the galK initiation codon remained the first AUG in the transcription unit. Using synthetic linkers, we then inserted specific sequences, each containing a single AUG codon at a defined position within the leader region. We found that the AUG inserts had widely different effects on galK expression and that the sequences surrounding the inserted AUG codons determined the magnitude of these effects. In addition, we placed these upstream inserted AUG codons into each of the three possible translation reading frames so that translation occurring in these frames terminates prior to, within, or downstream of the galK initiation codon, respectively. Single-nucleotide frameshift mutations were also introduced into one of these constructs to shift upstream translation into the other two possible reading frames. Depending upon where upstream translation stopped relative to the galK initiation codon, we observed consistently different effects on galK expression. Our results show that an upstream AUG that interferes with downstream translation initiation exerts its greatest effect when it translates out-of-frame through the downstream initiator into the gene. If translation is stopped upstream of or within the initiator, an unexpectedly high level of expression from the downstream AUG is maintained.

The Escherichia coli galactokinase gene (galK) was inserted into a modified early region transcription unit of simian virus 40 (SV40) contained on a bacterial plasmid. Introduction of this pSVK vector into monkey, mouse, and hamster cell lines by transfection resulted in efficient expression of the bacterial galK gene. This expression was shown to be dependent upon fusion of the galK gene to the early promoter of SV40 and did not appear to require SV40 splice signals. Moreover, expression in these cells could be obtained either transiently, 24--72 hr after transfection, or continuously, after stable transformation. In particular, pSVK-dependent galK expression was obtained in a hamster cell line genetically deficient in galactokinase activity. Expression of the bacterial enzyme was shown to complement the galactosemic defect of these cells, thereby allowing their selective survival and growth on galactose as the only carbon source. The ability to readily assay, select for, and potentially select against galK expression from pSVK and its derivatives should prove extremely useful in studying eukaryotic gene regulatory signals.

The mitochondrial isoenzyme of aspartate amino-transferase (mAspAT; subunit Mr 45,000) is synthesized on free polysomes in the cytosol as a precursor of higher Mr (pre-mAspAT; Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). We have isolated three overlapping cDNA clones that correspond almost to the full length of pre-mAspAT mRNA with 100 nucleotides at the 5‘ end missing. The mRNA is 2.1 kilobase pairs long and has a 3‘ noncoding region of 0.7 kilobase pairs. The cDNAs code for the 401 amino acid residues of mAspAT plus an NH2-terminal pre-piece. Deviations from the reported amino acid sequence were found at positions 154 and 202 where the cDNA specifies Gln instead of Glu. The pre-piece consists of 22 amino acid residues, among them 4 arginine and no acidic residues.

The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other.

The transcription initiation sites of the deooperon of Escherichia coliK12 have been determined in vitroand in vivo. Initiation of transcription occurs at two, tandemly arranged, homologous regions which are located 600 base pairs apart from each other. Evidence for a novel regulation mechanism of deogene expression involving the deoR repressor is presented.

Post‐transcriptional regulation of histone gene expression in a mouse mastocytoma cell cycle mutant (21‐Tb) depends largely on conserved DNA sequences that are essential for RNA 3′ processing. We have analyzed whether this regulation occurs at the level of RNA 3′ processing. We show, by RNase mapping, that nuclear H4 mRNA precursors, which are hardly detectable in total RNA from exponentially dividing cells, accumulate in G1‐arrested cells, i.e. when mature mRNAs are drastically reduced. Furthermore, we show that a heat‐labile component of the processing apparatus, recently identified in HeLa cell nuclear extracts, is limiting in extracts from G1‐arrested 21‐Tb cells. In contrast, this activity is in excess in extracts from exponentially dividing cells, whereas both extracts contain similar amounts of snRNPs of the Sm serotype. These fluctuations in the heat‐labile activity may generally contribute to proliferation or cell cycle dependent histone gene regulation.

We have analyzed the expression of endogenous histone H4 genes and of a newly introduced H4 gene in 21-Tb cells, a mouse mastocytoma cell-cycle mutant. Endogenous H4 mRNAs were less abundant by a factor of 120-180 in G1-arrested than in exponentially multiplying cells. However, H4 transcription rates were only decreased by a factor of 3 under these conditions, as determined by in vitro elongation of nascent transcripts. This indicates that post-transcriptional control of histone mRNA levels is important, in accord with published data. We introduced a mouse H4 gene, modified by a 12-base-pair (bp) insertion in its coding sequence, into 21-Tb cells by DNA-mediated gene transfer. The levels of transcripts from this gene were regulated in parallel with those of the endogenous genes. Moreover, fusion of the simian virus 40 (SV40) early promoter to a 463-bp fragment containing the 3'-terminal half of the mouse H4 gene, including 230 bp of spacer sequences, led to the regulated expression of SV40/H4 fusion RNA. However, a small proportion of SV40-initiated transcripts were not processed to histone-specific 3' ends, but extended farther through the downstream Escherichia coli galactokinase gene to a SV40 polyadenylylation site. In contrast to the short SV40/H4 RNA, the levels of these longer transcripts were not reduced in G1-arrested cells. These results show that sequences in the 3'-terminal part of the H4 gene can regulate gene expression in the cell cycle, presumably at the post-transcriptional level, as long as they are not positioned much more distant from the terminus than normal.

Infection of HeLa-cell monolayer cultures with rabbit poxvirus induces a marked decrease in cell-associated protein and in the activities of lactate dehydrogenase, acid phosphatase, and β-glucuronidase. This effect begins to occur around 10 hours post-infection (p.i.) and is accompanied by a concomitant rise of these enzyme activities in the culture medium. Only few cells detach from infected monolayers and these cannot account for protein release. Virion release can be inhibited at 4° C, whereas protein release cannot and it seems therefore that these events do not happen by a common mechanism. Moreover, penetration studies with [14C]-sucrose indicate that protein release reflects a true increase in plasma membrane permeability. Using the Gomori stain for acid phosphatase, a release of the enzyme into the cytoplasm around 8 hours p.i. can be confirmed rendering a causative role of lysosomal hydrolases in the pathogenesis of the observed plasma membrane permeability changes possible but not proving it.

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.