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The sporulation gene spoVK of Bacillus subtilis was cloned by use of the insertional mutation spoVK::Tn917 omega HU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK.
The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of specific regions, such as knobs, of individual maize chromosomes. A DNA hybridization blot panel of eight individual maize chromosome addition lines revealed that 180-bp repeats found in knobs are present in each of these maize chromosomes, but the copy number varies from approximately 100 to 25, 000. Cosmid clones with knob DNA segments were isolated from a genomic library of an oat-maize chromosome 9 addition line with the help of the 180-bp knob-associated repeated DNA sequence used as a probe. Cloned knob DNA segments revealed a complex organization in which blocks of tandemly arranged 180-bp repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. Sequence microheterogeneity including point mutations and duplications was found in copies of 180-bp repeats. The 180-bp repeats within an array all had the same polarity. Restriction maps constructed for 23 cloned knob DNA fragments revealed the positions of polymorphic sites and sites of integration of insertion elements. Discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes.
The thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of vaccinia virus has previously been mapped near the middle of the viral DNA, within the 4.85-kilobase HindIII J fragment, and shown to encode a Mr 19,000 polypeptide [Hruby, D. E. & Ball, L. A. (1982) J. Virol. 43, 403-409]. To locate the gene more precisely and to determine the structure of the basic transcriptional unit, the positions of cleavage sites for several restriction endonucleases were mapped within the HindIII J DNA fragment. Four appropriate subfragments of HindIII J DNA were inserted into plasmid pBR322 derivatives and cloned in Escherichia coli. These recombinant plasmid DNAs were tested for their ability to inhibit the cell-free synthesis of active thymidine kinase and to retain the mRNA for this enzyme when immobilized on nitrocellulose filters. The data showed that the gene spanned an EcoRI cleavage site that lies 850 base pairs from the left-hand end of the HindIII J fragment (the HindIII L-J boundary). Because hybridization of vaccinia virus DNA to partially purified thymidine kinase mRNA detected only a single 670-nucleotide RNA species capable of hybridizing to this region of the genome, nuclease S1 mapping experiments were carried out with thymidine kinase mRNA to protect DNA fragments that were terminally labeled at this EcoRI site. The results indicated that the gene extended from about 550 to 1,150 base pairs from the left end of HindIII J, was transcribed in a rightward direction, and contained no intervening sequences. Hence, a 1.04-kilobase Ava II-Hpa II restriction fragment containing this region of DNA was isolated and subjected to nucleotide sequence analysis. An examination of this nucleotide sequence revealed the presence of an open reading frame of 531 nucleotides capable of encoding a protein of 177 amino acids with a Mr of 20,077.
The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants. The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues that when altered lead to increased pump activity group together in two regions of the C-terminus. One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases. Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins.
Phages T2 and T4 induce synthesis of a DNA-adenine methylase which is coded for by a phage gene, dam+. These enzymes methylate adenine residues in specific sequences which include G-A-T-C, the methylation site of the host Escherichia coli dam+ methylase. Methylation of G-A-T-C to G-m6A-T-C protects the site against cleavage by the MboI restriction nuclease. We have taken advantage of this property to enrich and screen for transformants which contain a cloned, functional T4 dam+ gene. These recombinant molecules consist of a 1.85-kb HindIII fragment inserted into the plasmid pBR322; both orientations of the fragment express the methylase gene, suggesting that transcription is from a T4 promoter. We have tested the 1.85-kb insert for sensitivity to a variety of restriction nucleases and have found single sites for EcoRI, BalI, XbaI, and at least two sites for BstNI (EcoRII). The relative positions of these restriction sites have also been determined. Physical mapping was carried out by Southern blot hybridization with 32P-labeled (nick-translated clone) probe. These experiments showed that the insert corresponds to a HindIII fragment located on the physical map of T4 between positions 16.2 and 18.1 kb from the T4rIIA-rIIB junction. E. coli dam- possesses several phenotypic differences from the wild-type dam+ parent, including an increased sensitivity to 2-aminopurine (2-AP). We found that T4 dam+ clones could relieve dam- cells of their increased sensitivity to 2-AP.
We report the construction of an approximately 1.7-Mb sequence-ready YAC/BAC clone contig of 8p22-p23. This chromosomal region has been associated with frequent loss of heterozygosity (LOH) in breast, ovarian, prostate, head and neck, and liver cancer. We first constructed a meiotic linkage map for 8p to resolve previously reported conflicting map orders from the literature. The target region containing a putative tumor suppressor gene was defined by allelotyping 65 cases of sporadic ductal carcinoma in situ with 18 polymorphic markers from 8p. The minimal region of loss encompassed the interval between D8S520 and D8S261, and one tumor had loss of D8S550 only. We chose to begin physical mapping of this minimal LOH region by concentrating on the distal end, which includes D8S550. A fine-structure radiation hybrid map for the region that extends from D8S520 (distal) to D8S1759 (proximal) was prepared, followed by construction of a single, integrated YAC/BAC contig for the interval. The approximately 1730-kb contig consists of 13 YACs and 27 BACs. Fifty-four sequence-tagged sites (STSs) developed from BAC insert end-sequences and 11 expressed sequence tags were localized within the contig by STS content mapping. In addition, four unique cDNA clones from the region were isolated and fully sequenced. This integrated YAC/BAC resource provides the starting point for transcription mapping, genomic sequencing, and positional cloning of this region.
Restriction maps of genomes of the lytic form and diverse temperate mutants of phage theta of Bacillus licheniformis were constructed. Most temperate mutants produced fragmentation patterns identical to that of the parent lytic form, theta c: in other mutants the only detectable change in the map was the deletion of a Bg/II restriction endonuclease site at 46.5% genome length. In the genomes of two other temperate mutants, theta 1 and theta 2, the central part of the genome was replaced by a piece of DNA of equal length, but with a different distribution of restriction sites; the maps of the two mutants failed to reveal any similarity in the location of restriction sites in the inserted DNA. It seems that any alteration comprising the locus around the coordinate 46.5% of the theta c genome, brings about a transition from the lytic to temperate phenotype, indicating the position of a regulatory gene responsible for positive control of phage replication.
The flamenco gene, located at 20A1-3 in the beta-heterochromatin of the Drosophila X chromosome, is a major regulator of the gypsy/mdg4 endogenous retrovirus. As a first step to characterize this gene, approximately 100 kb of genomic DNA flanking a P-element-induced mutation of flamenco was isolated. This DNA is located in a sequencing gap of the Celera Genomics project, i.e., one of those parts of the genome in which the "shotgun" sequence could not be assembled, probably because it contains long stretches of repetitive DNA, especially on the proximal side of the P insertion point. Deficiency mapping indicated that sequences required for the normal flamenco function are located >130 kb proximal to the insertion site. The distal part of the cloned DNA does, nevertheless, contain several unique sequences, including at least four different transcription units. Dip1, the closest one to the P-element insertion point, might be a good candidate for a gypsy regulator, since it putatively encodes a nuclear protein containing two double-stranded RNA-binding domains. However, transgenes containing dip1 genomic DNA were not able to rescue flamenco mutant flies. The possible nature of the missing flamenco sequences is discussed.
A physical map of the genome of Rhizobium meliloti 1021 is presented. The physical sizes of the three replicons in this genome had previously been determined and are as follows: the chromosome, 3.4 Mb; pSym-b, 1.7 Mb; and pSym-a, 1.4 Mb. The physical maps for this GC-rich genome contain AT-rich restriction sites for SwaI (5'-TAAATTTA-3'), PacI (5'-TTAATTAA-3'), PmeI (5'-GTTTAAAC-3'), and, for pSym-b, SpeI (5'-ACTAGT-3'). In addition, the endonuclease I-CeuI cleaved the 23S rRNA genes in this genome, and perhaps in most eubacterial genomes. I-CeuI digestion and polymerase chain reaction amplification of rrn regions were used to determine that there are at least three rrn loci in R. meliloti, all of which are located on the chromosome. The orientation of the rrn loci was determined by Southern blotting with probes from rrn sequences located 5' and 3' to the I-CeuI site. The rrn loci are clustered in one part of the chromosome and are oriented so that transcription will occur away from a single point in the circle, as observed for the origin of replication in the Escherichia coli and Salmonella typhimurium chromosomes. Fifteen genes that had been tagged by Tn5 insertion were localized to fragments on the chromosome physical map by using the IS50 as a probe in Southern blots. In addition, glt and gap were placed on the physical map by using Southern hybridization with cloned genes. The fortuitous occurrence of SpecI site in Tn5-233 was used to physically map 10 genetically mapped Tn5-233 integrations on pSym-b and to anchor the physical map to the genetic map. Finally, we demonstrate the usefulness of the map by localizing a total of 12 previously unmapped transposon insertions in the genome. This is the first physical map of the genome of a multireplicon member of the family Rhizobiaceae as well as the first physical map of a Rhizobium chromosome.
To identify genes that contribute to myeloid leukemogenesis we have cloned viral integration sites from a CasBrM-MuLV-induced interleukin 3-independent myeloid leukemia cell line. Genomic probes derived from cellular sequences flanking two integrated proviruses were used to screen restriction digests of DNAs from a panel of 52 hematopoietic cell lines, 30 of which were established from CasBrM-MuLV- or MoMuLV-induced mouse leukemias. Probes from one integration site (His-1) defined a region that was rearranged in 3/52 cell lines, and probes from a second integration site (His-2) identified a rearrangement in 2/52 cell lines. Both cases of His-2 rearrangements occurred in concert with viral insertions in the His-1 locus. Genetic mapping of these loci using interspecific backcross analysis assigned the His-1 locus to mouse chromosome 2 and the His-2 locus to mouse chromosome 19. In situ hybridization with a probe from the human homologous region mapped the His-1 locus to human chromosome 2q14-q21. No recombinants were observed between His-2 and Gin-1, a common site of provirus integration in Gross passage A MuLV-induced T-cell leukemias, in 131 backcross animals, suggesting that these loci are tightly linked. The His-1 locus maps to mouse chromosome 2 distinct from any known oncogene or common site of integration but near the proximal breakpoint for a deletion that is observed in over 90% of radiation-induced leukemias.
Ten Staphylococcus aureus mutants, defective in the starvation-induced stationary phase of growth were isolated from two independent Tn917-LTV1 transposon insertion libraries and were designated suv as they had apparent survival defects. Seven of these mutants were defective under amino-acid-limiting conditions alone. Two mutants (suv-3 and suv-20) demonstrated lower plating efficiency when starved for glucose, phosphate or amino acids and one mutant (suv-11) had reduced plating efficiency after amino acid or glucose starvation. All of the mutants tested were as resistant to hydrogen peroxide assault as the parent, but six were more sensitive to low pH conditions. All the mutants were physically mapped on the S. aureus chromosome using PFGE. Chromosomal DNA flanking the Tn917-LTV1 insertion sites was rescued by cloning into Escherichia coli. DNA sequence analysis resulted in the identification of a number of transposon-disrupted ORFs encoding putative components such as superoxide dismutase (suv-1), haem A synthase (suv-3), a component of the SOS response (suv-9) and hypoxanthine-guanine phosphoribosyltransferase (suv-20). The Tn917-LTV1 insertion created lacZ transcriptional fusions for some of the stationary-phase loci. Expression analysis indicated that suv-4 was induced at mid-exponential phase, whereas suv-3 and suv-11 were induced at the onset of stationary phase. The possible roles of these suv components in stationary-phase survival or recovery is discussed.
INTRODUCTION: Cesium chloride has widely been used in experimental models to produce various ventricular arrhythmias. The study was designed to evaluate whether type and mechanism of these arrhythmias are dose-dependent. METHODS: In 7 dogs with acute AV-block, 60 pins containing 4 bipolar electrodes each were inserted into both ventricles to provide 240 endo-, epi- and midmyocardial recording sites. A computerized mapping system was used to determine three-dimensional activation patterns of ventricular arrhythmias induced by three injections of 1 mmol/kg cesium chloride at 20 minute intervals. RESULTS: Out of all arrhythmias induced, 25 ventricular extrasystoles, 31 monomorphic and 47 polymorphic ventricular tachycardias were mapped. Nonsustained ventricular tachycardias were readily inducible by a single bolus of cesium chloride, whereas sustained episodes required repetitive injections (1.45 +/- 0.61 vs. 2.61 +/- 0.57 doses, p < 0.05). Polymorphic tachycardias were observed more commonly than monomorphic tachycardias (87 vs. 31). Initiation and maintenance of cesium induced arrhythmias were exclusively based on focal mechanisms originating from the subendocardium, irrespective of morphology and dosage. All monomorphic arrhythmias were caused by repetitive firing of single immobile foci located in either the right or the left ventricle. Bi- and multifocal mechanisms, however, were found to underlie the polymorphic episodes. CONCLUSIONS: Although there is a dose-dependence as to the sustenance of mono- or polymorphic tachycardias, this does not reflect on the three-dimensional activation pattern of cesium induced arrhythmias, which are due to mono- or multifocal activation originating from the subendocardium.
Pigeon alpha-globin cDNA, previously cloned into the PstI site of pBR322 to yield pGP22 (Scobeleva et al., 1981), was mapped and sequenced. The insert was found to have an unusual structural organization, with 33 bp corresponding to the 5'-untranslated region of mRNA located roughly at the middle and flanked downstream by the 81 triplets coding for the N-terminal half of the alpha-globin chain. Moreover, 25 triplets identical to the 3'-terminal region of the insert were located upstream from the 5'-untranslated region and were followed by the coding sequence of 82 to 134 triplets. The last seven codons from 135 to 142 triplets were missing. The structural rearrangements in the cloned cDNA are discussed. The nucleotide sequence of alpha-globin mRNA from anemic pigeon was determined; it differs remarkably from those for chicken and duck, although homologies can easily be found. The predicted amino acid sequence of pigeon anemic alpha-globin is given.
Several vectors based on the widely used phage M13 and plasmid pUC were constructed. The vectors contain polylinkers (MCS) for DNA insertions with several new restriction sites (e.g., ApaI, NotI, StuI, SacII). Moreover, the NarI site in the nonessential part of the vector molecule was changed into a BssHII site, so that the NarI site in the MCS became unique.
Chagas disease and leishmaniasis are tropical diseases caused by the protozoan parasites, Trypanosoma cruzi and Leishmania species, respectively. Protein farnesyltransferase (PFT) is being investigated as a target for anti-trypanosomatid agents because inhibitors of this enzyme are highly toxic to these parasites compared to mammalian cells. Here, we report the cloning of the alpha- and beta-subunit genes of PFT from T. cruzi and Leishmania major. The proteins encoded by these genes are considerably larger than those of mammalian PFTs due to the presence of a number of inserts of >25 amino acids that map to junctions between helical structural elements. These inserts are not part of the active site or the interface between the two subunits. Northern blots demonstrate expression of messenger RNA for the PFT subunits in both mammalian and insect life-cycle stages of these parasites. The T. cruzi, Trypanosoma brucei, and L. major PFTs were overexpressed in the Sf9 cell/baculovirus system as active enzyme forms. Kinetic studies with a panel of CALX-containing peptides with all 20 amino acids in the X-position show that trypanosomatid PFTs have similar substrate specificities and these are different from the mammalian PFT substrate specificity patterns.
Electron microscopic analysis of in vitro transcriptional complexes of pBR322 and pACYC184 revealed five and six major transcriptional units, respectively, in these two plasmid vectors. These units are transcribed with various efficiencies, depending upon the individual promoter strengths, which differ in pBR322 up to 10-fold. A most interesting signal arrangement was found at the beginning of the tetracycline resistance region, where two partially overlapping promoters (P1 and P2) initiate transcription crosswise in opposite directions. Whereas P2 is known to promote tetracycline resistance and to be inactivated by HindIII cleavage, P1 is able to transcribe DNA integrated at that site and probably contributes to the expression of the beta-lactamase gene in pBR322. In pACYC184, besides P1, P2, and the cat (chloramphenicol resistance) promoter (P5), two initiation sites (P3 and P4) were mapped in a region that appears to be part of insertion sequence 1. The maps of transcription signals permit a more predictable utilization of these cloning vehicles and also allow the reinterpretation of earlier cloning results.
Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.
The S. rimosus amplifying sequence AUD-Sr1 encodes kanamycin and neomycin resistance, defined in the case of neomycin by aminoglycoside phosphotransferase. Its cloning on plasmid SLP1.2 makes possible the co-amplification of the obtained hybrid plasmids in S. lividans. In our study the regions responsible for resistance to aminoglycoside antibiotics and the capacity for amplification the two hybrid plasmids pSU10 and pSU3 were determined. Experiments on subcloning of the AUD-Sr1 sequence fragments on vector pIJ702 revealed localization of kanamycin and neomycin resistance determinants between PvuII(6) and BglII(7) on the AUD-Sr1 sequence fragments of 2.0 kb length. Two regions responsible for amplification of the hybrid plasmids were detected with deletion and insertion mapping. The first region is localized in the region of the plasmid SLP1.2 BamHI site and the second region is localized on the PstI(4)-PvuII(6) of the AUD-Sr1 sequence fragment of 1.1 kb length.