Splicing of yeast tRNA precursors: structure of the reaction intermediates.

The intermediates of the yeast tRNA splicing reaction have been characterized. The intervening sequence is excised as an unique linear molecule. It has 5'-hydroxyl and 3'-phosphate termini. Correspondingly, the half-tRNA molecules are shown to have a 3'-phosphate terminus on the 5' half and 5'-hydroxyl terminus on the 3' half. These isolated halves have been shown to be active in the ligation step of tRNA splicing. Removal of the 3'-phosphate from the 5' half eliminates the ability of the 5' half to participate in ligation.     

Restriction endonuclease BsoFI is sensitive to the 5'-methylation of deoxycytidines in its recognition sequence.

The isoschizomeric restriction endonucleases Fnu4HI and BsoFI cleave DNA at 5'-GCdecreasesNGC-3' sequences. Fnu4HI has been shown to be inhibited by 5'-CG-3'methylation in the sequences 5'-GmCGGC-3' or 5'-GCGGmCG-3'. We have now investigated the methylation sensitivity of BsoFI by testing its activity on plasmid DNA 5'-CG-3' methylated with the M.SssI DNA methyltransferase or on synthetic (CGG)n repetitive oligodeoxyribonucleotides which have been partly or completely C methylated. The data demonstrate that BsoFI cannot cleave at its recognition sequence when it is completely 5'-CG-3' methylated. These enzymes have proven to be useful in analyses of the methylation status in (CGG)n repeats of the human genome.     

Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction.

We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.     

Structure of a cluster of mouse histone genes.

The four mouse histone genes (2 H3 genes, an H2b gene and an H2a gene) present in a cloned 12.9 kilobase fragment of DNA have been completely sequenced including both 5' and 3' flanking regions. These genes are expressed in cultured mouse cells and the 3' and 5' ends of the mRNA have been determined by S1 nuclease mapping. These genes code for a minor fraction of the histone mRNAs expressed in cultured mouse cells. They comprise at most 5-8% of the total histone mRNA of each type. The two H3 genes code for H3.2 and H3.1 histone proteins, while the H2b gene codes for an H2b.1 protein with a single amino acid change (val-leu) at position 18. Only the 3' portion of the H2a gene is contained in the clone and there is an amino acid change (alanine-proline) at position 126. Comparison of the 5' and 3' flanking sequences reveals a conserved sequence at the 3' end of the mRNA which forms a hairpin loop structure. The codon usage in the genes is non-random and there has been no discrimination against CG doublets in the coding region of the genes.     

The human immunodeficiency virus type 1 Rev protein and the Rev-responsive element counteract the effect of an inhibitory 5' splice site in a 3' untranslated region.

A 5' splice site located in a 3' untranslated region (3'UTR) has been shown previously to inhibit gene expression. Natural examples of inhibitory 5' splice sites have been identified in the late 3'UTRs of papillomaviruses and are thought to inhibit viral late gene expression at early stages of the viral life cycle. In this study, we demonstrate that the interaction of the human immunodeficiency virus type 1 Rev protein with the Rev-responsive element (RRE) overcomes the inhibitory effects of a 5' splice site located within a 3'UTR. This was studied by using both a bovine papillomavirus type 1 L1 cDNA expression vector and a chloramphenicol acetyltransferase expression vector containing a 5' splice site in the 3'UTR. In both systems, coexpression of Rev enhanced cytoplasmic expression from vectors containing the RRE even when the RRE and the inhibitory 5' splice site were separated by up to 1,000 nucleotides. In addition, multiple copies of a 5' splice site in a 3'UTR were shown to act synergistically, and this effect could also be moderated by the interaction of Rev and the RRE. These studies provide additional evidence that at least one mechanism of Rev action is through interactions with the splicing machinery. We have previously shown that base pairing between the U1 small nuclear RNA and a 3'UTR 5' splice site is required for inhibition of gene expression. However, experiments by J. Kjems and P. A. Sharp (J. Virol. 67:4769-4776, 1993) have suggested that Rev acts on spliceosome assembly at a stage after binding of the U1 small nuclear ribonucleoprotein to the 5' splice site. This finding suggests that binding of additional small nuclear ribonucleoproteins, as well as other splicing factors, may be necessary for the inhibitory action of a 3'UTR 5' splice site. These data also suggest that expression of the papillomavirus late genes in terminally differentiated keratinocytes can be regulated by a viral or cellular Rev-like activity.     

Aminonucleosides and their derivatives. IV. Synthesis of the 3'-amino-3'-deoxynucleoside 5'-phosphates.

A new procedure has been developed for the synthesis of 3'-amino-3'-deoxyribonucleosides of adenine, cytosine and uracil by condensing the trimethylsilylated bases with peracylated 3-azido-3-deoxyribose derivative. The azido group could subsequently be reduced to amino. The 5'-phosphates of these nucleosides have been prepared and the analogues have been tested for their ability to stimulate the ribosome-catalyzed reaction of 3'(2')-O-(N-formylmethionyl) adenosine 5'-phosphate with phenylalanyl-tRNA.     

Terminal sequences of vesicular stomatitis virus RNA are both complementary and conserved.

The nucleotide sequences at the 5' and 3' termini of RNA isolated from the New Jersey serotype of vesicular stomatitis virus [vsV(NJ)] and two of its defective interfering (DI) particles have been determined. The sequence differs from that previously demonstrated for the RNA from the Indiana serotype of VSV at only 1 of the first 17 positions from the 3' terminus and at only 2 of the first 17 positions from the 5' terminus. The 5'-terminal sequence of VSV(NJ) RNA is the complement of the 3'-terminal sequence, and duplexes which are 20 bases long and contain the 3' and 5' termini have been isolated from this RNA. The RNAs isolated from DI particles of VSV(NJ) have the same base sequences as do the RNAs from the parental virus. These results are in sharp contrast to those obtained with the Indiana serotype of VSV and its DI particles, in which the 3'-terminal sequences differ in 3 positions within the first 17. However, with both serotypes, the 3'-terminal sequence of the DI RNA is the complement of the 5'-terminal sequence of the RNA from the infectious virus. These findings suggest that the 3' and 5' RNA termini are highly conserved in both serotypes and that the 3' terminus of DI RNA is ultimately derived by copying the 5' end of the VSV genome, as recently proposed (D. Kolakofsky, M. Leppert, and L. Kort, in B. W. J. Mahy and R. D. Barry, ed., Negative-Strand Virus and the Host Cell, 1977; M. Leppert, L. Kort, and D. Kolakofsky, Cell 12:539-552, 1977; A. S. Huang, Bacteriol. Rev. 41:811-8218 1977).     

Evolution of the casein multigene family: conserved sequences in the 5'' flanking and exon regions.

The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.     

Ab initio study of the reaction mechanism of ribonuclease A with cytidyl-3',5'-adenosine. I. Geometry optimization of cytidyl-3', 5'-adenosine.

As a first part of the ab initio study of the reaction mechanism of ribonuclease A with cytidyl-3',5'-adenosine, the geometry of the cytidyl-3',5'-adenosine substrate has been optimized using the Hartree-Fock method. Eleven different starting structures of cytidyl-3',5'-adenosine have been studied. To guarantee a proper alignment with the active site of the ribonuclease A enzyme, a part of the substrate was fixed during the geometry optimization. The geometry and intramolecular interactions of the refined conformations have been evaluated and two possible prototype structures have been proposed. One of these prototypes is more in accordance with the results of a molecular dynamics simulation and is therefore presented as a model for the geometry of cytidyl-3', 5'-adenosine in the initial step of the reaction with ribonuclease A.     

Biochemical effects of pure isomers of hexachlorobiphenyl: fatty livers and cell structure.

The effects of a single oral dose (50 mg/kg body wt.) of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 2,4,5,2',4',5'-HCB or 2,3,5,2',3',5'-HCB for a period of 72 h have been studied in the male rat. Only 3,4,5,3',4',5'-HCB caused necrosis of thymocytes. 3,4,5,3',4',5'-HCB caused marked pathological changes in the liver with less marked effects being caused by 2,4,5,2',4',5'-HCB and 2,3,5,2',3',5'-HCB. Total lipid content was increased by all the isomers studied, but 3,4,5,3',4',5'-HCB had more pronounced effect on total lipid content. Lipid accumulation was pericentral in the livers obtained from rats treated with 3,4,5,3',4',5'-HCB, but midzonal in the liver obtained from the rats treated with the other two isomers. Analysis of various lipid fractions showed that triacylglycerols were increased seven-fold only by 3,4,5,3',4',5'-HCB, while phospholipids were increased slightly by 2,4,5,2',4',5'-HCB or 2,3,5,2',3',5'-HCB. Only 3,4,5,3',4',5'-HCB increased the level of total and esterified cholesterol. These results show that the fatty livers caused by 3,4,5,3',4',5'-HCB were qualitatively and quantitatively different from those caused by the other two isomers at the same dose. For the first time a hexachlorobiphenyl unchlorinated in the para position, 2,3,5,2',3',5'-HCB has been shown to be a specific inducer of cytochrome P-450. The effects of 2,3,5,2',3',5'-HCB on cell structure and phospholipid content were quantitatively similar to those caused by 2,4,5,2',4',5'-HCB. Thus, chlorination at para (4,4') positions in chlorobiphenyls is not necessarily required for biological activity. It is hypothesized that net stereoelectronic properties of the isomers or resistance to metabolism may be the underlying factor in determining structure-activity relationships.     

Alterations of RNase H sensitivity of the 3'' splice site region during the in vitro splicing reaction.

We have developed a splicing assay system with an immobilized pre-mRNA to study the mechanism of the splicing reaction after spliceosome assembly. Using this system, we have found that the second step of the splicing reaction could be dissected into two stages. After the 5' splice site reaction, at least two factors interact with the pre-formed spliceosome containing intermediate molecules in an ATP-independent manner to convert the spliceosome into a form competent for the 3' splice site reaction. Then, the 3' splice site reaction occurs on this spliceosome, if ATP is supplied to the reaction mixture. We have also investigated the dynamic state of the 3' splice site region in the spliceosomes during the splicing reaction by probing with RNase H sensitivity. Prior to the 5' splice site reaction, the 3' splice site region was protected from RNase H attack. The region became sensitive immediately after the 5' splice site reaction, and subsequently became resistant again as the spliceosome competent for the 3' splice site reaction was formed. These results suggest that the interaction of the 3' splice site region with some spliceosome components changes significantly during the splicing reaction.     

Detailed structural analysis of two spliced HSV-1 immediate-early mRNAs.

The structures of two HSV-1 immediate-early mRNAs have been determined by nuclease-digestion procedures using 5' and 3' end-labelled DNA probes. These mRNAs, which map across the junctions between the short unique (US) and short repeat (IRS and TRS) genome regions, have common 5' portions located in IRS and TRS. The 3' portions, which extend into opposite ends of US, and unique. The DNA sequence encoding the common 5' portions largely comprises a 247 base pair (bp) leader region and a single intron of variable size. The variation in intron length is due to different copy numbers of a 22 bp tandem reiteration. A small proportion of the mRNA population is unspliced, but otherwise is identical to the more abundant spliced species.     

Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium.     

Synthesis and enzymic activity of 8-acyl and 8-alkyl derivatives of guanosine 3, 5-cyclic phosphate.

Several new 8-alkyl and 8-acyl derivatives of quanosie 3',5'-cyclic phosphate (cGMP) and inosine 3',5'-cyclic phosphate (cGMP) were prepared by direct alkylation or acylation of the parent cyclic nucleotide via free radicals generated in situ. These compounds have been examined for their ability to stimulate a cGMP-dependent protein kinase, and several of the cGMP derivatives were as active in this regard as cGMP. These compounds proved to be quite ineffective when tested for their ability to activate an adenosine 3',5'-cyclic phosphate (cAMP) dependent protein kinase. In addition, these 8-substituted cGMP derivatives are not substrates for a phosphodiesterase preparation from rabbit kidney, but do show inhibition of the hydrolysis of cAMP by crude phosphodiesterase preparations from rabbit lung and beef heart.