Studies on polylysogens containing lambda N-cI- prophages. II. Role of high multiplicities in lysogen formation by lambda N-cI- phage

Results of the experiments presented in this paper show that lambda N-cI- phage can lysogenize a nonpermissive host Escherichia coli when it infects at very high multiplicities (around 100), and lambda N-cI-cII- and lambda cIII-N-cI- lysogenize poorly at similar high multiplicities. The latter two phages lysogenize with appreciable frequency when either lambda N-cI- or lambda int-cN-cI-cII- is used as helper. The phages, lambda N-cI-, lambda N-cI-cII-, and lambda cIII-N-cI- can lysogenize also at relatively low m.o.i. of 20 in presence of the above lambda int-c helper, and the lambda int-cN-cI-cII- phage alone forms converted lysogens at an m.o.i. as low as 12. All these results suggest that the establishment of prophage integration by lambda N-cI- is positively regulated, like lambda N+cI+ phage, by the cII/cIII-promoted expression of the int gene of lambda, and under the N- condition, high multiplicities are needed to provide optimum levels of cII and cIII products, especially the latter.     

The superimmunity gene sim of bacteriophage P1 causes superinfection exclusion

Previous work has shown that the sim gene of bacteriophage P1, if cloned into a multicopy vector, confers immunity against P1 infection to cells. We show that a 1.85-kb DNA fragment from the sim region of P1 (in the multicopy plasmid pMK4) expresses immunity and encodes three proteins with molecular weights of about 25, 24, and 15 kDa. Deletion of 650 bp from the sim region abolished synthesis of all three proteins and of the sim phenotype. Expression of sim did not prevent adsorption of P1 to cells. Successful transfection with linear P1 DNA suggests that the recombinational circularization of P1 DNA is not inhibited in the presence of sim. Plasmid pMK4 and a P1 prophage can be stably maintained in the cell indicating that replication of the prophage is not disturbed by sim. The prophage can be induced in the presence of sim. This shows that the sim phenotype is not caused by preventing lytic replication or phage maturation. In cells with pMK4 there is no expression of genes from infecting phages and transduction frequency is drastically reduced. We suggest that sim functions as a superinfection exclusion system by preventing transfer of DNA from the adsorbed phages into the cytoplasm.     

Utilization of RNA polymerase I promoter and terminator sequences to develop a DNA transfection system for the study of hepatitis C virus internal ribosomal entry site-dependent translation.

BACKGROUND: Hepatitis C virus (HCV) causes severe liver diseases in a large population worldwide. HCV protein translation is controlled by an internal ribosomal entry site (IRES) within the 5'-untranslated region (UTR). HCV IRES-dependent translation is critical for HCV-associated pathogenesis. OBJECTIVE: To develop a plasmid DNA transfection system by using RNA polymerase I promoter and terminator sequences for studying HCV IRES-dependent translation. STUDY DESIGN: A gene cassette containing HCV 5'-UTR, Renilla luciferase reporter gene, and HCV 3'-UTR was inserted between RNA polymerase I promoter and terminator sequences. HCV IRES-directed translation was determined by luciferase assay after transfection. RESULTS: Transfection of the RNA polymerase I-HCV IRES plasmid into human hepatoma Huh-7 and HepG2 cells resulted in luciferase gene expression. Deletion of the IIIf domain in HCV IRES dramatically reduced luciferase activity. CONCLUSION: Our results indicated that the plasmid vector system-based on RNA polymerase I promoter and terminator sequences represents an effective approach for the study of HCV IRES-dependent translation.     

介导RNAi的tRNAVal启动子质粒载体的建立

目的 构建以tRNA^Val启动子控制shRNA转录引发RNAi的质粒载体。方法 从人基因组中用PCR方法扩增出tRNA^Val基因片段，人工突变去除3′末端数个碱基，连以Linker序列，用此经过改造的tRNA^Val启动子，构建控制针对荧光素酶的shRNA转录载体pUC-tRNA^Val lueRi，与pMAMneoLuc共转染BHK-21细胞，观察对荧光素酶表达的影响。结果 pUC-tRNA^Val lucRi可以高效特异性抑制pMAMneoLuc中荧光素酶的表达，效率达97．1％～99．5％。结论 tRNA^Val启动子载体可以高效转录shRNA，引发哺乳动物细胞RNAi现象。     

The 3' Igkappa enhancer contains RNA polymerase II promoters: implications for endogenous and transgenic kappa gene expression

We have created a kappa transgene in which a polymerase (pol) III promoter replaces the pol II promoter. Two independent transgenic lines show somatic hypermutation of the transgene in B cells from hyperimmunized mice. Both lines transcribe transgenes from the pol III promoter in the liver. However, in spleen and spleen B cell-derived hybridomas, they also transcribe mRNA from pol II promoters located within the 3' kappa enhancer of the preceding transgene copy in a tandem transgene array. The findings demonstrate that in an array of multiple transgenes the expression (and somatic hypermutation) of an individual transgene copy must be considered in the context of the other copies. We also show that sequences around the 3' kappa enhancer in endogenous genes are transcribed. The possible role of these promoters in endogenous kappa gene expression is discussed. An unrelated finding in this study was a novel RNA splice in one hybridoma.     

An evolutionarily conserved RNA stem-loop functions as a sensor that directs feedback regulation of RNase E gene expression

RNase E is a key regulatory enzyme that controls the principal pathway for mRNA degradation in Escherichia coli. The cellular concentration of this endonuclease is governed by a feedback mechanism in which RNase E tightly regulates its own synthesis. Autoregulation is mediated in cis by the 361-nucleotide 5' untranslated region (UTR) of rne (RNase E) mRNA. Here we report the determination of the secondary structure of the rne 5' UTR by phylogenetic comparison and chemical alkylation, together with dissection studies to identify the 5' UTR element that mediates autoregulation. Our findings reveal that the structure and function of the rne 5' UTRs are evolutionarily well conserved despite extensive sequence divergence. Within the rne 5' UTRs are multiple RNA secondary structure elements, two of which function in cis to mediate feedback regulation of rne gene expression. The more potent of these two elements is a stem-loop structure containing an internal loop whose sequence is the most highly conserved of any region of the rne 5' UTR. Our data show that this stem-loop functions as a sensor of cellular RNase E activity that directs autoregulation by modulating the degradation rate of rne mRNA in response to changes in RNase E activity.     

Transfection of Escherichia coli spheroplasts: infectious lambda prophage DNA.

High mol. wt. DNA was extracted from Escherichia coli lambda lysogens and was shown to be infectious. Its infectivity was due to prophage DNA integrated into the host chromosome rather than to DNA released from mature phage particles, as established by the following criteria: the titre of infectious DNA exceeded by 100-fold the titre of infectious units present before DNA extraction; mild shear selectively reduced prophage DNA infectivity to 2% of the unsheared DNA while lambda phage DNA infectivity retained 50% of its infectivity; DNA extracted from an E. coli (lambda c857 tsxisam6) lysogen yielded 200 times as many plaques on sup+ than on sup- spheroplasts. Thus lambda prophage DNA infectivity depends on expression of the excision gene while the infectivity of non-integrated forms of lambda does not. About 10(4) genome equivalents of E. coli DNA yielded one infectious centre unit in this assay system; this high infectivity should make prophage DNA a useful marker in genetic transformation experiments.     

Cloning and expression in Escherichia coli of opc, the gene for an unusual class 5 outer membrane protein from Neisseria meningitidis (meningococci/surface antigen).

A genomic library was constructed in a lambda gt11 vector using chromosomal DNA from a meningococcal serogroup A strain and plaques expressing the class 5C protein were recognized by screening with specific monoclonal antibodies. The opc insert was subcloned into a multicopy plasmid which induced expression of that protein in Escherichia coli as a surface-exposed major outer membrane protein. The nucleotide sequence of opc is typical of an outer membrane protein with a promoter and terminator region, a leader peptide which is cleaved during expression and a complete open reading frame. Unlike other meningococcal class 5 proteins or gonococcal P.II proteins, the sequence did not contain any pentanucleotide repeats and the sequence showed little homology to these other functionally related proteins. However, the predicted amino acid sequence of the mature protein for opc showed 27% similarity to that for a second opa gene cloned from the same meningococcal strain. This is the first report of cloning and expression of a functional meningococcal gene encoding a class 5 outer membrane protein in E. coli.     

Mitochondrial ribonuclease P activity of Trypanosoma brucei

Ribonuclease P (RNase P) is an essential enzyme that cleaves the 5' leader sequences of precursor tRNAs (pre-tRNAs) to generate mature tRNAs. The RNase P-like activity from Trypanosoma brucei mitochondria (mtRNase P) was purified over 10000-fold by sequential column chromatography. This is the first demonstration of such activity from mitochondria of parasitic protozoa. Its apparent molecular weight is approximately 70 kDa, considerably less than bacterial RNase P. Preliminary characterizations revealed no RNA component that is essential for this activity. Like other RNase P activities, the cleavage generates mature tRNAs with a terminal 5'-phosphate at the cleavage site and the 5' leader sequence with a 3'-hydroxyl. Disruption of the pre-tRNA tertiary structure inhibits the cleavage of the substrates. These data suggest that although all mitochondrial tRNAs are encoded in nuclear DNA in T. brucei, these cells contain an RNase P in the mitochondrion that cleaves the 5' terminal leader sequences of pre-tRNAs to generate mature tRNAs. Cleavage by mtRNase P of a pre-tRNA substrate that was divided into two fragments was demonstrated. This shows the feasibility of artificial regulation of gene expression that can be achieved by creating a complex made of target mRNA and a complementary small oligonucleotide that resembles natural substrates for RNase P.