Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.     

青霉素G酰化酶操纵子的负调控因子的研究

青霉素G酰化酶(PA)操纵子的调节基因(pacR)存在于青霉素G酰化酶结构基因(pac)内部Dral-Taql一段约500bp的DNA片段内,此片段内含有2个ORF。2个ORF及其突变体分别克隆到pUC18得到一系列重组质粒,用这些重组质粒转化青霉素G酰化酶产生菌E.coliD816,测定克隆片段对PA表达的影响。如果克隆片段含有具功能的pacR,诱导剂苯乙酸(PAA)不能使由高拷贝却pacR表达的阻抑物全部失活,部分阻抑物结合pac操纵基因,阻碍RNA聚合酶对pac的转录,因此PA的表达量降低。结果表明,阻抑物是由pac结构基因内部的ORF2编码的蛋白因子,pacR即ORF2。RNA—DNA杂交实验证实了pacR在转录水平阻抑pac的表达。     

Definition of the minimal cis-acting sequences necessary for genome maturation of the herpesvirus murine cytomegalovirus

Herpesvirus DNA replication proceeds via concatemeric replicative intermediates that are comprised of head-to-tail-linked genomes. Genome maturation is carried out by the terminase, a protein complex that mediates both insertion of concatemer DNA into capsids and its subsequent cleavage to release genomes within these capsids. This cleavage is sequence specific, but the governing cis-acting DNA sequences are only partially characterized. Two highly conserved motifs called pac1 and pac2 lie near the ends of herpesvirus genomes and are known to be critical for genome maturation. However, the potential importance of other sequences has not been fully investigated. We have undertaken to define all of the sequences necessary for efficient genome maturation for a herpesvirus by inserting ectopic cleavage sites into the murine cytomegalovirus genome and assessing their ability to mediate genome maturation. A combination of deletion and substitution mutations revealed that the minimal cleavage site is large (~180 bp) and complex. Sequences distal of pac1 (relative to the point of cleavage) were dispensable, suggesting that pac1 may be the sole cis-acting element on this side of the cleavage site. In contrast, a region distal to pac2 up to 150 bp from the point of cleavage was essential. Scanning substitutions revealed that the pac2 side of the cleavage site is complex and may contain multiple cis-acting sequence elements in addition to pac2. These results should facilitate the identification of trans-acting factors that bind to these elements and the elucidation of their functions. Such information will be critical for understanding the molecular basis of this complex process.     

The Gene Product of Human Cytomegalovirus Open Reading Frame UL56 Binds the pac Motif and Has Specific Nuclease Activity

Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5′-TAAAAA-3′ (pac 1) and 5′-TTTTAT-3′ (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults (2). For example, transplant and AIDS patients may develop life-threatening diseases as a consequence of primary infection or reactivation of latent infection. Present therapeutic approaches are limited, and new strategies that may result from a better understanding of the molecular events involved in viral maturation are needed.The HCMV virion consists of an envelope, an amorphous tegument, and an icosahedral nucleocapsid, which is assembled in the nuclei of infected cells. The precise molecular events of HCMV capsid assembly and subsequent DNA packaging are not well understood. It is generally accepted that viral DNA is packaged into a procapsid consisting of major capsid protein (UL86), minor capsid protein (UL85), minor capsid protein-binding protein (UL46), smallest capsid protein (UL47/48), assembly protein (UL80.5), and proteinase precursor protein (UL80a) (8). The assembly protein is removed during DNA insertion. It is unclear how the concatenated viral DNA contacts empty capsids and is cleaved and packaged into the capsid.Recent studies with herpes simplex virus type 1 (HSV-1) mutants that were temperature sensitive suggest that cleavage of the concatenated DNA does not occur in the absence of packaging (1). One possible model would be the involvement of cleavage packaging protein(s) which could facilitate incorporation of DNA into the procapsid by attaching to a specific motif within the viral genome. With HSV-1, the UL36 gene product (ICP1) and a smaller protein (possibly encoded by UL37) are part of a complex that recognizes the HSV-specific a sequence and are required for cleavage and packaging of viral DNA from concatemers (6, 7). In addition, the HSV-1 ICP 18.5 (UL28) gene product and the pseudorabies virus (PrV) homolog (16) were also reported to play an important role in DNA packaging (1, 14). Addison et al. (1) demonstrated that empty capsids were observed under conditions nonpermissive for the expression of the HSV-1 ICP 18.5 gene product. The HSV-1 ICP 18.5 mutants failed to cleave concatenated viral DNA in noncomplementing cells, suggesting that cleavage and packaging require ICP 18.5. Similar results were reported by Mettenleiter et al. (14) for PrV mutant protein. These observations suggest that the HSV-1 UL36, UL37, and UL28 gene products are involved in cleavage and packaging of concatenated viral DNA.In a recent study, we identified and partially characterized the gene product of HCMV UL56 (4). The HCMV UL56 gene product of 130 kDa is the homolog of the HSV-1 UL28 gene product. It is therefore postulated that UL56 possesses properties comparable to those of HSV-1 UL28, implying an involvement in cleavage and packaging of DNA. The HCMV genomic a sequence is a short sequence located at both termini of the genome and repeated in an inverted orientation at the L-S junction. The a sequence plays a key role in replication as a cis-acting signal for cleavage and packaging of progeny viral DNA and circularization of the viral genome. The HCMV a sequence contains two conserved motifs, pac 1 and pac 2, which are required for cleavage and packaging of the viral DNA (18). Both sequence motifs are located on one side of the cleavage site. The pac 1 and pac 2 motifs have an AT-rich core flanked by a GC-rich sequence. During the initial step of viral DNA packaging, a capsid-associated protein may bind to the pac sequences and may be involved in cleavage of the viral DNA concatemer.In this study, electrophoretic mobility shift assays (EMSAs) were performed with DNA probes spanning the region of these cis-acting elements. These studies demonstrate that specific proteins from HCMV-infected nuclear extracts or baculovirus-UL56-infected cell extracts bind to the pac motifs. Using affinity-purified monospecific antibodies, we show that p130 is present in specific DNA-protein complexes containing the pac motifs of the viral genome. Furthermore, evidence is presented for a sequence-specific endonuclease activity of recombinant HCMV p130, using circular plasmid DNA bearing the a sequence as a substrate.     

Recognition and cleavage of the bacteriophage P1 packaging site (pac). I. Differential processing of the cleaved ends in vivo

The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac. During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion. We show here that pac is located on a 620 base-pair fragment of P1 DNA (EcoRI-20). When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 "headfuls" (about 5 X 10(5) to 10 X 10(5) bases of DNA). Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage. Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for "late" protein synthesis (gene 10), do not affect pac cleavage. The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins. The products of pac cleavage were analyzed using two different DNA substrates. In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells. When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA. The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end. Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not. In a second case, pac was located in cells on a small (5 X 10(3) bases) multicopy plasmid. When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD- mutation. In recBCD- cells, the results with plasmid-pac substrates were similar to those obtained with chromosomally integrated pac substrates. We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.     

Cloning of a new FLO gene from the flocculating Saccharomyces cerevisiae IM1-8b strain

A flocculation conferring gene was cloned from a genomic library of the flocculating strain Saccharomyces cerevisiae IM1-8b as a 5 kb DNA fragment. The shortest DNA fragment (XbaI-XbaI) able to confer the flocculating phenotype was 3.1 kb. Southern analysis revealed that this gene was not homologous to the already reported FLO1 gene since strong hybridization signals were obtained when chromosomes IV and XII were probed with a digoxygenin-labelled fragment and no signal at all was detected for chromosome I. Partial sequencing data unequivocally ascribed the cloned fragment to chromosome XII. The gene was detected in a variety of S. cerevisiae strains regardless of their being phenotypically flocculating. This gene which, we propose as FLO2, is able to complement the flo1 mutation and is suppressed by suppressors (fsu3) that do not affect other FLO genes.     

Localization of Separate Genetic Loci for Reduced Sensitivity towards Small Isometric-Headed Bacteriophage sk1 and Prolate-Headed Bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2

The mechanism of reduced sensitivity to the small isometric-headed bacteriophage sk1 encoded on a 19-kilobase (kb) HpaII fragment subcloned from pKR223 of Lactococcus lactis subsp. lactis KR2 was examined. The reduced sensitivity to phage sk1 was due to a modest restriction/modification (R/M) system that was not active against prolate-headed phage c2. The genetic loci for the R/M system against sk1 and the abortive phage infection (Abi) mechanism effective against phage c2 were then localized by restriction mapping, subcloning, and deletion analysis. The restriction gene was localized to a region of a 2.7-kb EcoRV fragment and included an EcoRI site within that fragment. The modification gene was found to be physically separable from the restriction gene and was present on a 1.75-kb BstEII-XbaI fragment. The genetic locus for the Abi phenotype against phage c2 was localized to a region containing a 1.3-kb EcoRI fragment. Attempts to clone the c2 Abi mechanism independent of the sk1 R/M system were unsuccessful, suggesting that expression of the abi genes required sequences upstream of the modification gene. Some pGBK17 (vector pGB301 plus a 19-kb HpaII insert fragment) transformants exhibited the R/M system against phage sk1 but lost the Abi mechanism against phage c2. These transformants contained a 1.2- to 1.3-kb insertion in the Abi region. The data identified genetic loci on a cloned 19-kb HpaII fragment responsible for restriction activity and for modification activity against a small isometric-headed phage and for Abi activity against prolate-headed phage c2. A putative insertion element was also found to inactivate the abi gene(s).     

Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was Isolated as an Intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd30₁. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPH1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex plants in two such constructs BSL 12–1 and BSL 12–3.     

Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts.

Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S. alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S. alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 1H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolase and prg1, whose deduced amino acid sequence is significantly similar to that of degT, a Bacillus stearothermophilus pleiotropic regulatory gene.     

The R1 gene for late blight resistance in early and late maturing potato cultivars

The method of polymerase chain reaction was used to amplify a fragment of the LZ-NBS-LRR receptor kinase gene R1; the gene was transferred into potato (Solanum tuberosum) from its wild-growing relative S. demissum and confers the race-specific recognition of the pathogen Phytophthora infestans. To verify this method as a test for the presence of the late blight resistance gene R1, the amplified genome fragment was cloned from the potato hybrid comprising the germplasm of S. demissum. The primary structure of this fragment, which corresponded to the receptor domain of kinase, did not practically differ from the matching sequence in S. demissum. In addition, the method was verified by scoring the set of plant differentials, wherein the presence of R1 was established with race-specific Phytophthora isolates. By screening 70 potato cultivars, we established a significant relationship between the presence of the gene R1 fragment and the phenotypic characters of late blight resistance and late maturity. This evidence supports the idea that R1 was introgressed from short-day S. demissum into potato plants together with some gene(s) conferring late transition to flowering.     

Cloning and characterization of the gene for Salmonella typhimurium serine hydroxymethyltransferase

A plasmid containing the glyA gene of Salmonella typhimurium LT2 was constructed in vitro using plasmid pACYC184 as the cloning vector and a λgt7-glyA transducing phage as the source of glyA DNA. The recombinant plasmid (pGS30) contains a 10-kb EcoRI insert fragment. Genetic and biochemical experiments established that the fragment contains a functional glyA gene. From plasmid pGS30 we subcloned a 4.4-kb SalI-EcoRI fragment containing the glyA gene and its neighboring regions (plasmid pGS38). The location and orientation of the glyA gene within the 4.4-kb insert fragment was determined in four ways: (1) comparison of the physical map of the 4.4-kb SalI-EcoRI fragment with the physical map of a 2.6-kb SalI-PvuII fragment that carries the Escherichia coli glyA gene; (2) deletion analysis; (3) transposon Tn5 insertional inactivation experiments; (4) deoxyribonucleic acid sequencing and comparison of the S. typhimurium DNA sequence with the E. coli DNA sequence. A presumptive glyA-encoded polypeptide of M_r 47000 was detected using plasmid pGS38 as template in a minicell system, but not when the glyA gene was inactivated by insertion of a Tn5 element.     

In Silico and Deletion Analysis of Upstream Promoter Fragment of S-Adenosyl Homocysteine Hydrolase (SAHH1) Gene of Arabidopsis Leads to the Identification of a Fragment Capable of Driving Gene Expression in Developing Seeds and Anthers

S-adenosyl homocysteine hydrolase (SAHH) is a key enzyme in methylation metabolism of eukaryotes. A 1585 by fragment upstream to ATG of SAHH1 gene, was fused with a promoter-less β-Glucuronidase (GUS) gene and mobilized into Arabidopsis by Agrobacterium-mediated floral transformation to generate transgenic Arabidopsis. This fragment was found to drive constitutive expression of GUS in T₂ progeny of transgenic Arabidopsis. In silico analysis of the promoter region of SAHH1 suggested the presence of several cis-regulatory motifs including seed-specific motifs as well as anther-specific motifs in the 376 by (upstream to TSS of SAHH1) promoter fragment. Based on the partial deletion analysis carried out in the promoter region of SAHH1 (At4gl3940) this 376 by promoter fragment was found to be capable of driving GUS expression in developing seeds and in some anthers/micros pores.     

RFLP-PCR analysis of the aroA gene as a taxonomic tool for the genus Aeromonas

The aroA gene has been identified as a target in screening for the presence of most Aeromonas species so far described by PCR. Synthetic oligonucleotide primers of 24 and 25 nucleotides were used by PCR assay to amplify a sequence of the aroA gene, which encodes 3-phosphoshikimate-1-carboxyvinyltransferase, a key enzyme of aromatic amino acids and folate biosynthetic pathway. A 1236-bp DNA fragment, representing most of the aroA gene, according to the nucleotide sequence of A. salmonicida, was amplified from all Aeromonas species tested, which represented most of the 14 hybridization groups. HaeII digestion of the 1236-bp fragment generated a restriction fragment length polymorphisms which could be used as a powerful tool for identification of aeromonads to the genus level.     

Isolation and Characterization of OsGSTL1 Promoter from Rice

OsGSTL1 gene was isolated from the rice genomic library. Semi-quantitative RT-PCR analysis demonstrated that the expression of the OsGSTL1 in rice was not induced by chlorsulfuron, ethylene, abscisic acid, salicylic acid, and methyl jasmonate. In order to investigate the cis-elements of OsGSTL1 promoter, the promoter regions with different lengths were fused to the β-glucuronidase (GUS) reporter gene. All constructs were transformed into onion epidermal cells or A. thaliana plants to detect the expression patterns. In onion epidermal cells, the 160 bp fragment and longer ones were functional for directing GUS expression. In transgenic A. thaliana, the 2?155 bp upstream region of OsGSTL1 gene directed the GUS expression only in cotyledon after germination, but not in the root of young seedlings. In the later seedling, the 2?155 bp upstream region of OsGSTL1 gene directed GUS expression in roots, stems, and leaves. However, the GUS gene directed by a 1?224 bp upstream fragment is expressed in all the checked tissues. These results suggest that the spatiotemporal expression response elements of OsGSTL1 existed in the 5′-upstream region between −2?155 and −1?224 bp.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.