Inactivation and reversion of multisite streptomycin resistance mutations of Pneumococcus

Summary Purified DNAs were prepared from pneumococcal strains bearing either a multisite or single site streptomycin resistance (str-r) marker. In addition, each DNA contained a single site erythromycin resistance marker (ery-r2) or a pair of closely linked ery-r markers (ery-r2 and ery-r6). These DNA preparations were subjected to inactivation by subcritical heating or nitrous acid. Regardless of the genetic size of the streptomycin resistance marker, it was inactivated in a manner identical to that of the ery-r2 marker and less rapidly than that of the linked pair of ery-r markers.Spontaneous reversions towards decreased resistance have been observed in cultures of multisite mutants. Genetic analysis of the revertants revealed that the multisite mutations had been replaced by mutations with altered properties of recombination.The simplest interpretation of the evidence is that these are point mutations of such a nature, or occurring in such a region, that recombination is inhibited in the region immediately adjacent to them. In this way they would appear genetically large but physically small.Supported by NSF grant GB-7295.     

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

Spline methods for the comparison of physical and genetic maps.

The first genetic maps were constructed by linkage analysis. Physical mapping techniques, such as radiation hybrids and complete sequencing, produce a different picture. For the purposes of population genetics, clinical genetics, and genetic epidemiology, it is important to harmonize and amalgamate existing genetic and physical maps. Among other things, comparisons of the two kinds of maps promotes better understanding of the wide variation in local recombination rates per unit physical length of DNA. The current paper presents methods for estimating recombination intensity as a function of physical distance along a chromosome. Genetic map distance is the integral of intensity. We derive fast reliable estimation algorithms based on a Poisson process model, penalized likelihoods, and cubic spline interpolation. Our methods provide a rigorous and statistically sound foundation for comparing physical and genetic maps. To illustrate the possibilities, we apply the methods to published recombination data on CEPH families and the complete sequences of chromosomes 21 and 22. Our results are in good agreement with previous studies and the biological data.     

High-resolution genetic and physical mapping of the Rar1 locus in barley

 The Mla-12-mediated resistance in barley against Erysiphe graminis f. sp. hordei requires for its function the Rar1 gene. High-resolution genetic mapping was accomplished by inspecting more than 4000 plants segregating for Rar1 within an 0.7-cM interval containing the target gene. Marker enrichment in the target region was carried out by an amplified fragment length polymorphism (AFLP)-based search for polymorphic loci using bulked DNA templates from resistant and susceptible recombinants adjacent to Rar1. RFLP markers closely linked to Rar1 were used to investigate the relationship between physical and genetical distances by PFGE Southern analysis, indicating the physical linkage of two genetically separated RFLP loci. Comparative mapping of Rar1-linked RFLP probes in barley and rice identified a break of collinearity in the orthologous chromosome segments. Received: 11 February 1998 / Accepted: 3 March 1998     

Study of MFD-type repair in locus determining resistance of Escherichia coli to streptomycin

The yield of induced mutations to streptomycin resistance (Str) in E. coli, UV-irradiated and temporarily incubated in liquid medium not permitting protein synthesis, depends upon the conditions of preirradiation growth and preirradiation treatment of the bacteria, i.e. on their physiological state at the moment of irradiation. This fact is not readily reconciled with a model postulating mutation production in the structural genes of E. coli during excision repair. A preferred explanation is offered, based on the assumption that the efficiency of mutagenesis at the rpsL (strA) locus is determined by interference of antimutagenic (generalized excision repair and MFD) and promutagenic (mutation fixation of excision repair) events. The participation of macromolecular syntheses in Str mutation fixation is suggested.     

High-resolution genetic map of the Lv resistance locus in tomato

 Bulked segregant analysis and high-resolution mapping were used to pinpoint the position of the Lv gene for resistance to Leveillula taurica in tomato. Mapping in an F₂, corresponding to more than 3800 gametes, indicates that Lv is positioned within the 0.84-cM interval defined by the RFLP markers CT121 and CT129, with the closest marker, CT121, being only 0.16 cM from the gene. The tight linkage of these markers demonstrates their usefulness in marker-assisted breeding for Lv, and the high-resolution map provides a starting a starting point for positional cloning of this resistance gene. Received: 25 February 1997 / Accepted: 21 March 1997     

Dihydrofolate reductase and aminopterin resistance in Pneumococcus

Summary Wild-type pneumococci derived from Avery's strain R36A are sensitive to extracellular concentrations of the folate antimetabolite aminopterin exceeding 1.0x10^-6 M. Three classes of resistant strains are phenotypically distinguishable: amiB-r, amiA-r and amiD-r strains are resistant to low (1.5x10^-6 M), intermediate (0.5–4.0×10^-5 M) and high (4.5x10^-4 M) aminopterin levels respectively. The amiA and amiB regions are weakly linked, but linkage has not been established between either of these loci and the amiD region.Consistent with the maximum resistance conferred by mutations in the amiA locus, dihydrofolate (FH₂) reductase in cell-free extracts (CFE) of amiA-r strains has a two- to six-fold greater affinity for the substrate than dose the enzyme in wild-type CFE (Table 1); FH₂ reductase from amiA-r strains may also have reduced affinity for aminopterin. Specific activity of the enzyme is not affected by mutation in the amiA locus (Table 1) and its affinity for the cofactor (NADPH) is probably unaffected by mutation in this locus (Table 4). Dihydrofolate reductase activity in amiA5 CFE is considerably more thermolabile than that in wild-type CFE (Table 2).The enzyme in CFE of the high resistance strain amiD1 has the same affinity for the substrate, cofactor and antimetabolite as FH₂ reductase in wild-type CFE (Figs. 1–4, 8 and 9; Table 4). However, specific activity of the enzyme in amiD1 CFE is 11-fold higher than that in wild-type CFE (Table 1) and it is much more heat stable (Table 2).Some properties of FH₂ reductase in CFE of the high resistance recombinant strain amiA5amiD1 are intermediate between those in CFE of wild-type and amiD1.Preliminary results suggest that CFE of wild-type and amiA5 contain a factor, which is neither dialyzable nor heat sensitive, that has an inhibitory effect upon activity and stability of FH₂ reductase in amiD1 CFE (Tables 2 and 3).     

High resolution genetic and physical mapping of the mouse asebia locus: A key gene locus for sebaceous gland differentiation

The asebia (ab) mutation in the mouse is an autosomal recessive trait with hypoplastic sebaceous glands. As a first step toward cloning the ab gene, we report here the genetic mapping of the ab locus with respect to Chromosome 19 microsatellite markers. 644 backcross progeny were generated by mating (CAST/EiJ × DBA/1LacJ-ab^2J/ab^2J) F₁ heterozygous females and parental ab^2J/ab^2J mutant males. Our results located the ab gene to an interval of 1.6 cM on mouse Chromosome 19 defined by flanking markers D19Mit11 and D19Mit53/D19Mit27, and identified a tightly linked polymorphic marker, D19Mit67, that co-segregates with the mutation in the backcross progeny examined. This places ab locus 4 cM distal to its present position as indicated in Mouse Genome Database at The Jackson Laboratory. We have also mapped a yeast artificial chromosome (YAC) contig in this locus interval which suggests the ab interval to be less than one megabase of DNA.     

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.     

Refined genetic and comparative physical mapping of the canine copper toxicosis locus

Recently, the copper toxicosis (CT) locus in Bedlington terriers was assigned to canine chromosome region CFA10q26, which is homologous to human chromosome region HSA2p13-21. A comparative map between CFA10q21-26 and HSA2p13-21 was constructed by using genes already localized to HSA2p13-21. A high-resolution radiation map of CFA10q21-26 was constructed to facilitate positional cloning of the CT gene. For this map, seven Type I and eleven Type II markers were mapped. Using homozygosity mapping, the CT locus could be confined to a 42.3 cR₃₀₀₀ region, between the FH2523 and C10.602 markers. On the basis of a partial BAC contig, it was estimated that 1-cR₃₀₀₀ is equivalent to approximately 210 kb, implying that the CT candidate region is therefore estimated to be about 9 Mb. Received: 16 December 1999 / Accepted: 23 February 2000     

Genetic and physical localization of the root-knot nematode resistance locus Mi in tomato

As part of a map-based cloning strategy designed to isolate the root-knot nematode resistance gene Mi, tomato F2 populations were analyzed in order to identify recombination points close to this economically important gene. A total of 21?089 F2 progeny plants were screened using morphological markers. An additional 1887 F2 were screened using PCR-based flanking markers. Fine-structure mapping of recombinants with newly developed AFLP markers, and RFLP markers derived from physically mapped cosmid subclones, localized Mi to a genomic region of about 550?kb. The low frequency of recombinants indicated that recombination was generally suppressed in these crosses and that crossovers were restricted to particular regions. To circumvent this problem, a population of Lycopersicon peruvianum, the species from which Mi was originally introgressed, that was segregating for resistance was developed. Screening of this population with PCR, RFLP and AFLP markers identified several plants with crossovers near Mi. Recombination frequency was approximately eight-fold higher in the Mi region of the L. peruvianum cross. However, even within the wild species cross, recombination sites were not uniformly distributed in the region. By combining data from the L.?esculentum and L. peruvianum recombinant analyses, it was possible to localize Mi to a region of the genome spanning less than 65?kb.     

An integrated physical, genetic and cytogenetic map around the sunn locus of Medicago truncatula.

The sunn mutation of Medicago truncatula is a single-gene mutation that confers a novel supernodulation phenotype in response to inoculation with Sinorhizobium meliloti. We took advantage of the publicly available codominant PCR markers, the high-density genetic map, and a linked cytogenetic map to define the physical and genetic region containing sunn. We determined that sunn is located at the bottom of linkage group 4, where a fine-structure genetic map was used to place the locus within a approximately 400-kb contig of bacterial artificial chromosome (BAC) clones. Genetic analyses of the sunn contig, as well as of a second, closely linked BAC contig designated NUM1, indicate that the physical to genetic distance within this chromosome region is in the range of 1000 -1100 kb.cM-1. The ratio of genetic to cytogenetic distance determined across the entire region is 0.3 cM.microm(-1). These estimates are in good agreement with the empirically determined value of approximately 300 kb.microm(-1) measured for the NUM1 contig. The assignment of sunn to a defined physical interval should provide a basis for sequencing and ultimately cloning the responsible gene.