Derivation-dependent distribution of insertion sites for a Drosophila transposon.
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Animals↗
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Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome.
In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.
Cloning, Molecular↗
Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis.
Linker insertion mutagenesis was employed to create structural disruptions of the lethal factor (LF) protein of anthrax toxin to map functional domains. A dodecameric linker was inserted at 17 blunt end restriction enzyme sites throughout the gene. Paired MluI restriction sites within the linker allowed the inserts to be reduced from four to two amino acids. Shuttle vectors containing the mutated genes were transformed into the avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the gene products. Mutations at five sites in the central one-third of the sequence made the protein unstable, and purified protein could not be obtained. Mutated LF proteins with insertions at the other sites were purified and assessed for toxic activity in a macrophage lysis assay and for their ability to bind to the protective antigen (PA) component of anthrax toxin, the receptor binding moiety. Most insertions located in the NH2-terminal one-third of the LF protein eliminated both toxicity and binding to PA, while all four insertions in the COOH-terminal one-third of the protein eliminated toxicity without affecting binding to PA. These data support the hypothesis that the NH2-terminal domain contains the structures required for binding to PA and the COOH-terminal domain contains the putative catalytic domain of LF.
Antigens, Bacterial↗
Mapping and identification of essential gene functions on the X chromosome of Drosophila.
The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.
Animals↗
DNA repair mutants of Rhodobacter sphaeroides.
The genome of the photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1 comprises two chromosomes and five endogenous plasmids and has a 65% G+C base composition. Because of these characteristics of genome architecture, as well as the physiological advantages that allow this organism to live in sunlight when in an anaerobic environment, the sensitivity of R. sphaeroides to UV radiation was compared with that of the more extensively studied bacterium Escherichia coli. R. sphaeroides was found to be more resistant, being killed at about 60% of the rate of E. coli. To begin to analyze the basis for this increased resistance, a derivative of R. sphaeroides, strain 2.4.1 delta S, which lacks the 42-kb plasmid, was mutagenized with a derivative of Tn5, and the transposon insertion mutants were screened for increased UV sensitivity (UVs). Eight UVs strains were isolated, and the insertion sites were determined by contour-clamped homogeneous electric field pulsed-field gel electrophoresis. These mapped to at least five different locations in chromosome I. Preliminary analysis suggested that these mutants were deficient in the repair of DNA damage. This was confirmed for three loci by DNA sequence analysis, which showed the insertions to be within genes homologous to uvrA, uvrB, and uvrC, the subunits of the nuclease responsible for excising UV damage.
Amino Acid Sequence↗
Amplification of a short nucleotide sequence in the repeat units of defective herpes simplex virus type 1 Angelotti DNA.
It has been shown earlier that the reiterated regions TRS and IRS bracketing the Us segment of herpes simplex virus type 1 Angelotti DNA are heterogeneous in size by stepwise insertion of one to six copies of a 550-base-pair nucleotide sequence. Considerably higher amplification of this sequence was observed in defective viral DNA: up to 14 copies were detected to be inserted in the repeat units of a major class of defective herpes simplex virus type 1 Angelotti DNA, dDNA1, which originated from noncontiguous sites located in UL and the inverted repeats of the S component of the parental genome. Physical maps were established for the cleavage sites of KpnI, PstI, XhoI, and BamHI restriction endonucleases on the repeats of dDNA1. The map position of the insertion sequence was determined. It was demonstrated that the amplified inserts were not distributed at random among or within the repeats. A given total population of dDNA1 molecules consisted of different homopolymers, each of which contained a constant number of inserts in all of its repeats. Assuming that a rolling-circle mechanism is involved in the generation of full-length defective herpes simplex virus type 1 Angelotti DNA from single repeat units, these data suggest that the 550-base-pair sequence is amplified in the repeats before the replication process.
DNA Replication↗
DNA cloned from the ayw subtype of hepatitis B virus.
DNA from the ayw subtype of hepatitis B virus (HBV) was ligated into the EcorI site of DNA from plasmid pBR322 and propagated in E coli chi 776. A plasmid with a 3200 base pair insert (pHBV-1) was isolated and the cloned HBV DNA was mapped with restriction endonucleases. Differences were found in restriction endonuclease cleavage sites for DNAs from HBV of subtype ayw and adr.
Chromosome Mapping↗
Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain.
Two gene transfer systems were established for a marine bacterium, Vibrio sp. strain 60. One was generalized transduction with a newly isolated bacteriophage, As3, and the other was conjugal gene transfer by the use of newly constructed transposon-facilitated recombination (Tfr) donors. As3 transduced various chromosomal markers at frequencies of 10(-4) to 10(-6). Tfr donors, which were constructed by introducing transposon Tn10 into both plasmid RP4 and the chromosome, mediated the polarized transfer of chromosomal genes from the sites of Tn10 insertion on the chromosome. By means of these gene transfer systems, a genetic map of the vibrio chromosome was constructed.
Bacteriophages↗
The effect of changing the distance between the TATA-box and cap site by up to three base pairs on the selection of the transcriptional start site of a cloned eukaryotic gene in vitro and in vivo.
We have studied how small changes in the distance between the TATA-box and cap site affect transcription of a eukaryotic gene in vitro and in vivo. The trout protamine gene TPG-3 [Gregory et al. (1982) Nucl. Acids Res. 10, 7581-7592] is a good model for such a study as it has (i) a consensus TATA-box 32 base pairs (bp) upstream from an A-residue which is the natural cap site (designated +1) (ii) two further A-residues at -5 and +5, providing alternative transcriptional start sites which are in significantly different sequence environments and (iii) a unique AvaII restriction site immediately downstream from the TATA-box which is ideal for the insertion or deletion of up to 3bp. Transcripts of the wild type and mutant genes were generated in vitro using a HeLa whole cell extract or 'in vivo' by transient expression following their transfection into HeLa cells. These 'spacer' mutations did not affect the efficiency of transcription of the gene in vitro but they did affect the selection of transcriptional start site both in vitro and 'in vivo'. Analysis of 5'-ends by S1-mapping and primer extension showed that the A-residue(s) selected are those which, by insertion or deletion, come to lie on the same face of the DNA double helix as the TATA-box, although the DNA sequence in the immediate vicinity of the potential start sites influences their utilisation. Comparison of the TPG-3 wild type transcripts in these experimental systems with natural mRNA suggests that cap site selection is more stringent in the developing trout testis.
Animals↗
Defining a common region of deletion at 13q21 in human cancers.
Previous molecular genetic analyses identified a region of deletion at 13q21 in a variety of human cancers, suggesting the existence of a tumor suppressor gene(s) at this locus. In our earlier study on prostate cancer, the region of deletion was confined to a 3.1 cM interval between D13S152 and D13S162. At present, however, no known gene located in this interval has been firmly implicated in cancer, and the region remains too large for gene identification. To fine-map the area of interest, we established a contig of bacterial artificial chromosome (BAC) clones, narrowed the region of deletion by loss of heterozygosity (LOH) and homozygosity-mapping-of-deletion (HOMOD) analyses in different types of cancers, and tested a candidate gene from the region for mutation and alteration of expression in prostate cancers. The contig consisted of 75 overlapping BAC clones. In addition to the generation of 47 new sequence-tagged-site (STS) markers from the ends of BAC inserts, 76 known STS and expressed sequence tag markers were mapped to the contig (25 kb per marker on average). The minimal region of deletion was further defined to be about 700 kb between markers D13S791 and D13S166 by LOH analysis of 42 cases of prostate cancer, and by HOMOD analysis of eight prostate cancer cell lines/xenografts and 49 cell lines from cancers of the breast, ovary, endometrium, and cervix, using 18 microsatellite markers encompassing the deletion region. A gene that is homologous to the WT1 tumor suppressor gene, AP-2rep (KLF12), was mapped in this region and was analyzed for its expression and genetic mutation. In addition to low levels of expression in both normal and neoplastic cells of the prostate, this gene did not have any mutations in a group of aggressive prostate cancers and cell lines/xenografts, as assessed by the methods of polymerase chain reaction-single strand conformational polymorphism analysis and direct sequencing. These studies suggest that a 700 kb interval at 13q21 harbors a tumor suppressor gene(s) that seems to be involved in multiple types of cancer, and that the AP-2rep gene is unlikely to be an important tumor suppressor gene in prostate cancer. The BAC contig and high-resolution physical map of the defined region of deletion should facilitate the cloning of a tumor suppressor gene(s) at 13q21.
Chromosome Deletion↗
Mapping of the ARS-like activity and transcription initiation sites in the non-canonical yeast mitochondrial ori 6 region.
The insert-containing, non-canonical ori 6 region of yeast mitochondrial DNA of Saccharomyces cerevisiae was dissected into 15 different segments that were ligated to the integrative yeast vector YIp5. Six recombinant plasmids exhibited replicative ability in yeast and carried consensus sequences similar to the previously described 11 bp motifs active as autonomous replication sequences (ARS). In addition, all active constructions carry one or more of the characteristic GC-rich domains A, B or C present in the ori 6 region, thus confirming and expanding the study of Blanc (Gene 30 (1984) 47-61) with the canonical ori 5. Also a new transcriptional origin is activated in the ori 6 region, apparently circumventing a disruption by insertion of a GC-rich sequence that, in this ori, removes the mitochondrial promoter usually present next to the C element. The ARS-positive constructions correspond to the retained segments of spontaneous well-characterized suppressive or neutral petite genomes that contain segments of the ori sequence.
Base Sequence↗
N-independent leftward transcription in coliphage lambda: deletions, insertions and new promoters bypassing termination functions.
Lambda mutants capable of N-independent red-gam gene expression were isolated by selecting Fec+ plaque-forming derivatives of lambda N+ nutL- (Fec-) strains. In addition to true nutL+ reversions, three classes of second-site mutations were identified: (1) ninL deletions that remove a region containing either tL1 or both tL1 and tL2 termination signals, or only a small region (defining the rut site) just upstream from tL1, (2) new constitutive promoters that map just upstream from the tL2 termination site and which are created either by point mutations (hip) or by short insertion sequences (isp), (3) small internal deletions in gene cro. The positions and individual effects of these mutations, some of which only partially abolish termination function, provide evidence for a complex multipartite structure of the termination signals.
Chromosome Mapping↗
Specialized transduction by bacteriophage P22 in Salmonella typhimurium: genetic and physical structure of the transducing genomes and the prophage attachment site.
P22pro-1 and P22pro-3 are specialized transducing derivatives of phage P22 that carry the proA and proB genes of Salmonella typhimurium. These genes lie immediately adjacent to the prophage attachment site on the bacterial chromosome. By examining DNA heteroduplexes in the electron microscope, we found that DNA molecules from P22pro-1 and P22pro-3 each contain a substitution which adds length to the composite genome making the intracellular replicated genome too long to fit into a single phage particle. In this respect, and in many of their biological properties, the proline-transducing phages resemble P22Tc-10, another specialized transducing phage with an oversize, intracellular replicated genome which carries a tetracycline-resistance determinant from an R-factor.--Unlike P22Tc-10, however, P22pro-1 and P22pro-3 fail to integrate normally during lysogenizing infections, even when provided with all known integration functions. These results suggest that the proline substitutions have created a defect in the phage attachment site and suggest that the Campbell model for the formation of specialized transducing phages is applicable to phage P22 with the additional feature that oversize genomes can be produced and propagated.--A physical and genetic map of the P22 genome near the prophage attachment site was constructed which shows that the insertion from the R-factor in P22Tc-10 is not at the attachment site: it is therefore unlikely that P22Tc-10 was formed in an abnormal prophage excision event as envisioned in the Campbell model, but was instead the result of a direct translocation from the R-plasmid to P22.
Chromosome Mapping↗
Nucleotide sequence, insertional mutagenesis, and transcriptional mapping of a conserved region of the baculovirus Autographa californica nuclear polyhedrosis virus (map unit 64.8-66.9).
The nucleotide sequence of a 2773-bp region of the Autographa californica nuclear polyhedrosis virus HR3 variant (AcMNPV) (map unit 64.4-68.2) has been determined. This region lies between the previously analyzed p6.9 and p80 genes. Three open reading frames were contained within this region, potentially coding for proteins of 45, 40, and 12 kDa. The 5' ends of transcripts capable of coding for the 45- and 12-kDa proteins map to consensus baculovirus late transcription start sites (ATAAG), while the major 5' end of the transcript coding for the 40-kDa protein maps 16 nucleotides downstream from another late transcription start site sequence, GTAAG. Attempts to prepare recombinant viruses containing insertions within these three genes were negative, suggesting that they are all essential for virus replication in cell culture. The size and organization of transcripts expressed from this region of AcMNPV were very similar to those of the previously described homologous region of Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV). The three AcMNPV protein sequences (p45, p40, and p12) showed amino acid sequence homology with the proteins p48, p45, and p12 of OpMNPV, suggesting this region is highly conserved in baculoviruses.
Amino Acid Sequence↗
Z DNA and loop structures by immunoelectronmicroscopy of supercoiled pRW751, a plasmid containing left-handed helices.
Single and multiple loops were seen when the plasmid pRW751 was allowed to react with anti-Z-DNA or with a Z-specific cross-linking agent. Loop formation was dependent upon negative supercoiling and the presence of Z-specific antibody or cross-linking agent. Restriction enzyme mapping located 18 sites at the bottoms of loops, in addition to the two (dG-dC)n inserts of pRW751. No more than 5 loops were seen in any of the measured molecules; thus, not all potential Z-sites assume the Z conformation at any particular time. Stretches of alternating purine-pyrimidine sequences occur at all 20 sites. Almost all of the Z sites could be mapped to regions located at the beginnings or ends of reading frames or at various regulatory sites. Our findings support the concept that supercoiling brings distant sequences to within 5A of one another, allowing joint participation in regulatory processes controlled by DNA-binding proteins.
DNA Restriction Enzymes↗
Site specificity of mutations arising in dysgenic hybrids of Drosophila melanogaster.
Mutations arising in dysgenic hybrids of Drosophila melanogaster were collected in the zeste-white region of the X chromosome. A preponderance of the mutations affected the zw1 locus; many of these were asociated with structural abnormalities including inversions, deficiencies, and insertions in the 3A3-4 region of the polytene chromosome map. The extreme sensitivity of the zw1 locus to the mutator activity of dysgenic hybrids contrasted with the apparent insensitivity of the zw2 locus. Other loci in the zeste-white region were weakly sensitive to the mutator activity. Insertions of two and six bands were seen between bands 3A4 and 3A6 in the chromosomes of one of the zw1 mutant stocks examined. Another insertion was detected at position 2F4-5 in a different stock. Many of the mutant chromosome were evidently unstable, as judged by secondary breakage in other parts of the X chromosome. The cytogenetic data are consistent with the idea that mutations arising in dysgenic hybrids are caused by transposable elements which insert preferentially at certain sites on the chromosomes.
Animals↗
cDNA clones for liver cytochrome P-450s from individual aroclor-treated rats: constitutive expression of a new P-450 gene related to phenobarbital-inducible forms.
Differential hybridization and screening with cloned inserts was used to identify two families of cytochrome P-450 cDNA clones in libraries prepared from total liver poly(A)+RNA of individual Aroclor-treated rats. One family has cDNA inserts for the major phenobarbital-inducible P-450s, P-450b and P-450e. Two types of P-450e inserts were identified. In addition, irregular inserts were characterized from two clones (PB23 and PB24) of this group. The other family has cDNA inserts for the major 3-methylcholanthrene-inducible species, P-450c and P-450d. No coding sequence restriction site variants were detected among 26 P-450d and P-450c inserts analyzed. The restriction map of the irregular 2.2-kb PB23 insert has a P-450b-like portion, followed by a 3' extension that hybridizes to RNAs of 2.7 and 4.8 kb, which are also detectable with a classical P-450b probe. The PB23 insert and the 2.7- and 4.8-kb RNAs presumably represent 3' extensions of P-450b/P-450e mRNAs, polyadenylated at downstream sites. The 858-bp sequence of the PB24 insert encodes the carboxy-terminal portion of a P-450b/P-450e-like protein. There is approximately 20% divergence at the polypeptide level between the PB24 and P-450b/P-450e sequences; nevertheless, they share many essential features. A PB24-specific probe hybridizes to a 1.9-kb RNA species which is present in the liver of untreated rats and which is not appreciably induced by phenobarbital or Aroclor. The PB24 cDNA most likely represents a constitutive cytochrome P-450, related to phenobarbital-inducible forms.
Amino Acid Sequence↗
Deletion analysis of essential genes of Escherichia coli: investigation of the btuB-rpoBC interval.
A method is described for deletion mapping of essential genes in Escherichia coli. It involves the isolation of secondary-site insertions of lambda cI857plac5 into an F' plasmid. The transposed lacZ gene is useful both for the ready screening of plasmid-phage cointegrates and for rapid analysis of deletions that extend from the site of phage integration into the bacterial genes carried on the substituted plasmid. Such deletions may be used to 'hook-up' bacterial cistrons to the powerful lac promoter. We report the application of this technique to the study of the btuB-rpoBC interval, a cluster of genes encoding components of the transcription-translation apparatus.
Bacteriophage lambda↗