Library-independent cloning of genomic fragments adjacent to vector integration sites: isolation of the EB4-PSV gene from a Dictyostelium gene disruption transformant.

We have designed an efficient procedure to clone genomic DNA adjacent to the integration site of transformation vectors. Using this method on a Dictyostelium gene disruption transformant, we have cloned a 5kb fragment which has previously escaped isolation by conventional library screening. Our protocol eliminates recloning of the original vectors which are often integrated in multiple tandem copies (1) but specifically recovers vectors containing genomic fragments adjacent to the integration site. The protocol is useful to isolate flanking fragments in gene disruption transformants to characterize flanking regions which may influence the expression of transformed genes by position effects and to identify sites of insertional mutagenesis.     

An improved vector system for insertional gene inactivation inspired by the tmRNA-tagging system of S. pneumoniae

Insertional mutagenesis is a technique often used to inactivate genes in Streptococcus pneumoniae. Using conventional vectors, a 5' segment of the targeted gene remains under the control of the gene's authentic promoter following gene disruption. Thus, the expression of a functional peptide and the misinterpretation of results in consequence cannot be excluded. To circumvent this problem, we have developed a plasmid for insertional mutagenesis based on the tmRNA-tagging system of S. pneumoniae which ensures that any protein expressed after gene disruption is degraded. Insertional mutagenesis using this vector results in the targeted gene being tagged with a tmRNA-derived sequence coding for a proteolysis tag. Here we show that the translation product of a gene tagged by this method is not detectable by Western blotting, suggesting that the protein was degraded. This modified vector allows total inactivation of genes with a reliability that cannot be achieved by conventional vectors for insertional mutagenesis. This approach can be applied to other bacterial species.     

Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells.

Eukaryotic expression vectors have been used successfully in viral LT-expressing cell lines (ie. COS) to clone cDNAs encoding proteins that can be detected through their bio-activity or reactivity with specific antibodies. Since Chinese hamster ovary cells (CHO) have been used extensively for the isolation and characterization of somatic cell mutants, we felt it would be an advantage to develop an expression cloning system in CHO cells. We have modified the eukaryotic expression vector CDM8 by replacing the polyoma and SV40 origins of replication with the 427bp non-coding region of the Syrian hamster papovavirus. Wild-type CHO cells and the CHO glycosylation-mutant Lec4A were transfected with plasmids bearing the early genes of either polyoma virus or hamster papovavirus in order to establish stable, LT antigen-expressing cell lines designated CHOP or CHOH, respectively. CHOP cell lines expressing polyoma LT antigen supported efficient replication of CDM8, but replicated pMH poorly. Conversely, CHOH cells expressing the hamster papovavirus LT antigen supported replication of pMH, and at a lower efficiency, CDM8. Replication of CDM8 and pMH vectors were equally efficient in selected CHOP and CHOH cell lines, respectively and comparable to that of CDM8 replication in COS-1 cells. A bacterial beta-galactosidase fusion gene inserted into the multiple cloning site of a CDM8 derivative was efficiently expressed when transiently transfected into CHOP and CHOH cells but not CHO cells since only the former supports autonomous plasmid replication. These results show that expression-cloning in CHO cells expressing either polyoma virus or hamster papovavirus LT antigens is possible using either the CDM8 or the pMH vectors, respectively.     

Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids.

The thymidine kinase (TK) gene of HSV-1 has been cloned in Escherichia coli K12 plasmids, pMH1, pMH1A, and pMH4. These plasmids contain a 1,92Obp HSV-1 TK DNA sequence, which replaces a 2,067 bp EcoR I to Pvu II sequence of plasmid pBR322 DNA. Superhelical DNAs of plasmids pMH1, pMH1A, and pMH4 as well as plasmid DNAs cleaved by EcoR I, Hinc II, Bg1 II, Sma I, and Pvu II transformed TK-deficient LM(TK-) cells to the TK+ phenotype. A 1,230bp EcoR I-Sma I fragment purified from pMH1 DNA (and from plasmid pAGO, DNA, the parent of pMH1) also transformed LM(TK-) cells. Serological and disc PAGE studies demonstrated that the TK activity expressed in biochemically transformed cells were HSV-1-specific. The experiments suggest that the HSV-1 TK coding region may be contained within a l.1kbp DNA sequence extending from about the Hinc II (or Bgl II) cleavage site to the Sma I site. 35S-methionine labeling experiments carried out on cell lines transformed by Hinc II-cleaved pMH1 DNA and by the EcoR I-Sma I fragment showed that the TKs purified from the transformed cells consisted of about 39-40,000 dalton polypeptides.     

Mutational alterations of translational coupling in the L11 ribosomal protein operon of Escherichia coli.

The L11 operon in Escherichia coli consists of the genes coding for ribosomal proteins L11 and L1. It is known that translation of L1 does not take place unless the preceding L11 cistron is translated, that is, the two cistrons are translationally coupled, and this is the basis of coregulation of the translation of the two cistrons by a single repressor, L1. Several mutational analyses were carried out to define the region responsible for coupling L1 translation with L11 translation. First, by introducing several amber mutations into the L11 gene by a site-directed mutagenesis technique, it was shown that translation by ribosomes down to a position 21 nucleotides upstream, but not to a position 45 nucleotides upstream, from the end of the L11 cistron allowed the initiation of L11 translation. Second, deletion analysis indicated that a region located 23 to 20 nucleotides from the end of the L11 gene was involved in preventing independent initiation from L1 translation. Third, five different mutations obtained by screening for activation of the masked L1 initiation site were found to be clustered in a small region immediately upstream from the Shine-Dalgarno sequence of L1, and all of them were G-to-A transitions. These results, together with some additional experiments with oligonucleotide-directed mutagenesis, defined the region involved in the coupling and suggest that some special feature of this region, probably different from simple masking of the initiation site by base pairing, is responsible for translational coupling. The present results also suggest that there might be specific differences in the primary nucleotide sequence that distinguish independent translational initiation sites from translationally coupled (i.e., masked) initiation sites.     

Characterization of the plasmids comprising the “R Factor” R5 and their relationships to other R plasmids

The “R factor” R5 confers resistance to tetracycline (Tc), streptomycin (Sm) and spectinomycin (Sp), chloramphenicol (Cm), sulfonamides (Su), kanamycin (Km), and mercuric ion (Mer). This phenotype is mediated by the presence of two R plasmids: pMH1 and pMH2, having approximate weights of 18.5 and 62 megadaltons (Mdal), respectively. pMH1 encodes Sm, Su, Cm, Mer, and Km resistance, and is nonconjugative. pMH2 confers Sm, Su, Cm, Mer, and Tc resistance, is conjugative, and belongs to the FII incompatibility group. NR79 is a 63-Mdal R plasmid encoding the same resistances as “R5,” and was derived from the same geographical source. It belongs to the FII incompatibility group and is conjugative. Analysis of restriction endonuclease digestion patterns and polynucleotide sequence homologies indicate that pMH1, pMH2, and NR79 are closely related. In addition, pMH2 and NR79 exhibit nearly complete homology to R100. Restriction endonuclease maps and resistance gene locations for pMH1, pMH2, and NR79 have been derived and a model for the evolutionary relationships of these plasmids is presented.     

RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells

Gene trapping is a form of insertional mutagenesis that causes disruption of gene function. Here we report the construction and extensive examination of a versatile retrovirus vector, RET (removable exon trap). The RET vector uses an improved poly A-trap strategy for the efficient identification of functional genes regardless of their expression status in target cells. A combination of a potentially very strong splice acceptor and an effective polyadenylation signal assures the complete disruption of the function of trapped genes. Inclusion of a promoterless GFP cDNA in the RET vector allows the expression pattern of the trapped gene to be easily monitored in living cells. Finally, because of loxP-containing LTRs at both ends, the integrated proviruses can be removed from the genome of infected cells by Cre-mediated homologous recombination. Hence, it is possible to attribute the mutant phenotype of gene-trapped cells directly to RET integration by inducing phenotypic reversion after provirus excision. The RET system can be used in conjunction with cell lines with functional heterozygosity, embryonic stem cells, lineage-committed cell lines that differentiate in response to specific inducing factors and other responsive cell lines that can be selected by virtue of their induced green fluorescence protein expression.     

Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei

Trypanosoma brucei variant surface glycoprotein (VSG) genes are activated either by duplicative (DA) transposition of the gene to a pre-activated expression site or by nonduplicative (NDA) activation of a previously silent telomeric gene. We have obtained a recombinant clone spanning the 5' barren region of the expression linked copy of the duplicated VSG gene 117a. By DNA sequence and hybridization analyses we have identified a pleomorphic family of 14-25 non-VSG genes that lie upstream of both DA and NDA VSG expression sites. These expression site associated genes (ESAGs) encode 1.2 kb poly(A)+ mRNAs that are specifically transcribed from the active VSG expression telomere in mammalian bloodstream stages of T. brucei but, in common with VSG genes, are not transcribed in procyclic culture forms. cDNA and genomic sequences predict open reading frames that are conserved in the two ESAGs examined.