RFLP analysis of chromosomal fragments in genetic mosaic strains of Bombyx mori

Mottled striped (p^Sm), a genetic mosaic strain for larval body marking of the silkworm, Bombyx mori, contains a small chromosomal fragment generated by breakage near the end of the 2nd chromosome. This fragment carries the striped marking (p ^S ) gene and part of the chorion gene clusters. To determine the structural features of this fragmented chromosome, we studied the organization of the chorion genes on the fragment using restriction fragment length polymorphism (RFLP) analysis. Tow of three classes of chorion gene family probes detected RFLPs in the two phenotypes, p^Sm (p/p/p ^S ), and p (p/p), which had lost the fragment, segregated among the siblings of a mottled striped strain. Although hybridization patterns were basically identical between them, one or two additional bands were always observed associating with the mosaic phenotype (p^Sm). This suggests that the additional bands correspond to extra copies of chorion genes on the chromosomal fragment, which have a different structure from ones on the intrinsic 2nd chromosomes. Such heterogeneity of chorion genes may have been maintained since the beginning of mosaic induction, due to the absence of recombinational events between the two chromosomes. We are unable to detect any RFLPs by hybridization with the early class of chorion genes, implying that chromosomal breakage might have occurred between the two chorion clusters, Chl-2 and Ch3, which are located approximately 4 cM apart from each other. Based on RFLP analyses for two independent mosaic strains (788 and 872), we postulate a common chromosomal origin with independent breakpoints and construct structural models for the two kinds of chromosomal fragments.     

Restriction and modification in Bacillus species: genetic transformation of bacteria with DNA from different species, part I.

Summary Host specific restriction was detected in 13 Bacillus strains, when 63 strains of Bacillus subtilis and 15 other Bacillus strains were tested with phage 105C. These 13 strains were classified into 8 groups (M,H,C,N,E,F,G,P) by the type of restriction. M-type strains (B. subtilis Marburg 168, its derivatives, and two other strains) showed relatively weak restriction, restricting 105C from other groups of Bacillus by ratios of 10^-1 to 10^-3. Strains of groups H,C,N,E,F,G, and P restricted 105C from other groups by ratios of 10^-2 to 10^-8. It was confirmed with some of the strains that type-specific modification was endowed only by the last host. Furthermore, we isolated one restriction deficient mutant of B. subtilis marburg 168-YS11, which had also lost its modification phenotype.     

Genetic structure and origin of t haplotypes of mice, analyzed with H-2 cDNA probes

We investigated the genetic organization and evolutionary origin of t chromosomes of mice by examining the restriction fragment patterns of DNA from t haplotypes and normal chromosomes with cDNA probes to H-2 class I genes. On genomic DNA blots, the restriction fragments containing H-2-related sequences were highly variable among different inbred strains of mice, whereas they were very similar among different t haplotypes even when the t haplotypes carried serologically different H-2 haplotypes. These observations suggest that all t haplotypes have a common origin and are not products of independent mutational events. We also mapped the position of several restriction fragments characteristic of t DNA by using a battery of recombinant t haplotypes, defined with respect to their t-lethal factors and H-2 haplotypes. We thus show that restriction fragments containing H-2-related sequences map to the left of the H-2 class I genes in t chromosomes, a region in which the tw32 b-lethal factor also maps. The cloning of these fragments can be expected to provide an entry for the structural analysis of t DNA.     

Cloning and in silico Mapping of Resistance Gene Analogues Isolated from Rice Lines Containing Known Genes for Blast Resistance

We amplified resistance gene analogues (RGAs) from the genomic DNA of 10 rice lines having varying degree of resistance to Magnaporthe grisea by using degenerate primers and various RGAs were mapped in silico on different rice chromosomes. The amplified products were grouped into 3–8 restriction fragment length polymorphic classes by using Mbo1 and Alu1 restriction enzymes. Of 98 RGAs obtained in this study, 65 RGA clones showed more than 95% homology with various RGAs sequences present in the GenBank. Phylogenetic analysis of these RGAs formed 11 groups. Using sequence homology approach, RGAs isolated in this study were physically mapped on 23 loci on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12. Twenty RGAs were mapped near to the chromosomal regions containing known genes/QTLs for rice blast, bacterial leaf blight and sheath blight resistance. Thirty‐nine RGA sequences also contained open reading frame representing signature of potential disease resistance genes.     

Rapid Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">acidophilus</Emphasis> by Restriction Analysis of the 16S–23S rRNA Intergenic Spacer Region and Flanking 23S rRNA Gene

A rapid molecular approach was developed for the initial identification of Lactobacillus acidophilus strains which are difficult to identify using a single biochemical test. The 16S–23S rRNA intergenic spacer regions and flanking 23S rRNA genes of 19 strains of lactobacilli were amplified and the nucleotide sequences and restriction site polymorphisms were analyzed. AluI was the most useful of the restriction enzymes analyzed and produced reproducible digestion profiles in the L. helveticus, L. plantarum, and L. casei groups, as well as in L. acidophilus. This restriction fragment length polymorphism method may be useful for the identification of L. acidophilus strains in dairy products.     

Chromosome assignment of four photosynthesis-related genes and their variability in wheat species

Copy numbers of four photosynthesis-related genes, PhyA, Ppc, RbcS and Lhcb1 ^*1, in wheat genomes were estimated by slot-blot analysis, and these genes were assigned to the chromosome arms of common wheat by Southern hybridization of DNA from an aneuploid series of the cultivar Chinese Spring. The copy number of PhyA was estimated to be one locus per haploid genome, and this gene was assigned to chromosomes 4AL, 4BS and 4DS. The Ppc gene showed a low copy number of small multigenes, and was located on the short arm of homoeologous group 3 chromosomes and the long arm of chromosomes of homoeologous group 7. RbcS consisted of a multigene family, with approximately 100 copies in the common wheat genome, and was located on the short arm of group 2 chromosomes and the long arm of group 5 chromosomes. Lhcb1 ^*1 also consisted of a multigene family with about 50 copies in common wheat. Only a limited number of restriction fragments (approximately 15%) were used to determine the locations of members of this family on the long arm of group 1 chromosomes owing to the multiplicity of DNA bands. The variability of hybridized bands with the four genes was less in polyploids, but was more in the case of multigene families. RFLP analysis of polyploid wheats and their presumed ancestors was carried out with probes of the oat PhyA gene, the maize Ppc gene, the wheat RbcS gene and the wheat Lhcb1 ^*1 gene. The RFLP patterns of common wheat most closely resembled those of T. Dicoccum (Emmer wheat), T. urartu (A genome), Ae. speltoides (S genome) and Ae. squarrosa (D genome). Diversification of genes in the wheat complex appear to have occurred mainly at the diploid level. Based on RFLP patterns, B and S genomes were clustered into two major groups. The fragment numbers per genome were reduced in proportion to the increase of ploidy level for all four genes, suggesting that some mechanism(s) might operate to restrict, and so keep to a minimum, the gene numbers in the polyploid genomes. However, the RbcS genes, located on 2BS, were more conserved (double dosage), indicating that the above mechanism(s) does not operate equally on individual genes.     

Three extra copies of a C4-related gene in H-2 w7 mice are C4/Slp hybrid genes generated by multiple recombinational events

Mice bearing the H-2 _w7 haplotype have five C4-related genes and constitutively express the Slp antigen. To understand the structure and evolution of the five C4-related genes of the C3H.W7 mouse, we have determined nucleotide sequences of the 5 end region of these genes. A C4/Slp hybrid nature was confirmed for three of five C4-related genes as predicted previously by restriction enzyme analysis. The nucleotide sequences of the 5 flanking regions of these three hybrid genes showed close similarity to that of the C4 gene, while the 3 side of the ninth exon of the three hybrid genes showed close similarity to that of the Slp gene. In contrast, the regions between the first exon and the middle of the ninth exon of the three hybrid genes showed a mosaic structure of C4-like and Slp-like sequences. Moreover, the boundaries of the C4-like and Slp-like sequences were quite different among the three hybrid genes. The pattern of nucleotide sequence diversity in this region among the five C4-related sequences could be mainly explained not by point mutations but by gene conversions or unequal crossovers. These results suggest that multiple genetic recombinational events between two homologous sequences played an important role in the generation and diversification of the extra copies of the C4/Slp gene in the H-2 ^w7 mouse.The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and have been assigned the accession numbers D90167-71.     

Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectria haematococca

Summary The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda⁺ isolates) hybridized to PdaT9, while only one Pda^– isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda^– phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6–2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation.     

Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles

The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed. The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp). Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G+C present at the site of restriction. EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested. Thus, the 41 strains fell into 30 restriction groups using only two enzymes. Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome. Correspondence to: C. Diviès     

Moused-amino-acid oxidase gene: Restriction fragment length polymorphism among mouse strains

Summary Genomic DNA was extracted from mice of 15 strains (A/J, AKR, BALB/c, C3H/He, C57BL/6, CBA/J, CD-1, CF#1, DBA/2, ddY/DAO⁺, ddY/DAO^–, ICR, NC, NZB and NZW) for the examination of the difference in the structure of thed-amino-acid oxidase gene among the mouse strains. The DNAs were digested with restriction endonucleases and analyzed by Southern hybridization usingd-amino-acid oxidase cDNA as a probe. The 15 strains showed the same hybridization patterns in theEcoRV,BamHI orBglII digestion. In theEcoRI digestion, the DBA/2 strain showed a different hybridization pattern from the other 14 strains. In thePvuII andXbaI digestion, C3H/He, CBA/J, ddY/DAO⁺ and NC strains were different from the other 11 strains. In thePstI andHindIII digestion, restriction fragment length polymorphisms were observed, and the 15 strains were classified into four groups according to their hybridization patterns. These results indicate that the 15 strains of mice carry a structurally similard-amino-acid oxidase gene, but there is a variation in its inside sequence among the groups of the strains.     

Genes coding for respiratory complexes map on all three chromosomes of the Paracoccus denitrificans genome

The genome of Paracoccus denitrificans (strain Pd1222) consists of three distinct DNA molecules when separated by standard pulsed-field gel electrophoresis with apparent molecular sizes of approximately 2, 1.1, and 0.64 Mb. When the separated chromosomes are digested by restriction enzymes and sizes of resulting fragments are summed up, the three chromosomes are composed of 1.83, 1.16, and 0.67 Mb. Since their migration behavior relative to size standards is largely independent of electrophoresis conditions, at least the two smaller chromosomes most likely represent linear molecules. The size analysis presented here allows an unequivocal distinction between groups of different strains of P. denitrificans and of Thiosphaera pantotropha, confirming an earlier cytochrome c analysis. When the genome was analyzed with different probes coding for respiratory enzymes, essential genes were found spread over all three chromosomes without any obvious clustering on any of the three forms. Received: 10 August 1997 / Accepted: 10 November 1997     

Chromosomal location and copy number in wheat and some of its close relatives of genes for enzymes involved in photosynthesis

Summary Homologous probes for the wheat coding sequences of the enzymes phosphoribulokinase, phosphoglycerate kinase (both chloroplast and cytosolic forms), chloroplast fructose-1,6-bisphosphatase and the small subunit of ribulose-1,5-bisphosphate carboxylase were used to determine the copy number and chromosomal location of the genes encoding these enzymes by restriction fragment length polymorphism analysis. Heterologous probes were similarly used to characterize the genes for the enzymes glyceraldehyde phosphate dehydrogenase (both chloroplast and cytosolic forms), phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase. Several of the genes are present in single copies per haploid genome, and the different enzymes are encoded by loci dispersed on different chromosomes. The significance of these findings is discussed in relation to gene expression and control of copy number.     

Localization of C4 genes within the HLA complex by molecular genotyping

Segregation of the complement component, C4, was analyzed in six families that each included an individual who inherited an HLA haplotype where a crossover event had occurred in the region between HLA-B and HLA-DR. Two cDNA clones corresponding to the C4 gene were utilized as probes in Southern blot analysis of DNA from members of each family. Restriction fragment length polymorphisms (RFLP) were observed and were assigned to haplotypes. In one family RFLP, hybridizing with the C4 probes, segregrated with HLA-B, and in four families RFLP segregated with HLA-DR; one family was not informative in this respect. These analyses have made it possible to localize the genes for C4 between HLA-B and HLA-DR by molecular genotyping and to characterize three different genomic configurations of C4 genes by limited restriction mapping.Abbreviations RFLP restriction fragment length polymorphisms - LCL lymphoblastoid cell lines     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

A genetic linkage map of the MSM Japanese wild mouse strain with restriction landmark genomic scanning (RLGS)

A high-resolution genetic map of the Mus musculus molossinus (MSM) Japanese wild mouse strain was constructed with restriction landmark genomic scanning (RLGS) and compared with that of the laboratory strain C3H. MSM is phylogenetically 1 million years apart from common laboratory mouse strains and is distinctly resistant to chemical carcinogenesis. Since it exhibits frequent genetic polymorphisms with laboratory mice but can still be easily crossed with laboratory strains, hybrids between MSM and carcinogen-sensitive laboratory mouse strains provide excellent materials for analysis of modifier genes and genetic changes during carcinogenesis. We have generated MSM backcross progeny with the C3H strain, which is extremely sensitive to hepatocarcinogenesis, to construct the present map. RLGS profiles with two combinations of restriction enzymes (NotI–PvuII–PstI, NotI–PstI–PvuII) yielded more than 2000 spots each. The polymorphism rate was about 39.2%, and of a total of 1732 polymorphic spot loci identified, 1371 could be assigned to specific chromosomes by comparison with 79 microsatellite marker loci. Thus, 1450 loci, on all chromosomes except for Y, effectively mapped 90% of the genome (1431.7 cM length). Although some spots might be derived from the same NotI site, each NotI site potentially generating two fragments, the presence of at least 515 loci groups with different progeny distribution patterns dispersed through the genome with an average spacing of 3 cM, means that this genetic map should be useful for analysis of various biological phenomena, including carcinogenesis and ontogenesis, at the gene level. Received: 25 August 1999 / Accepted: 20 December 1999     

GENETIC CHARACTERIZATION OF STRAINS OF CYANOBACTERIA USING PCR-RFLP OF THE cpcBA INTERGENIC SPACER AND FLANKING REGIONS1

Oligonucleotide primers, specific for conserved regions of the genes encoding the β- and α-phycocyanin subunits of phycobilisomes (cpcB and cpcA) of cyanobacteria, were used to amplify a DNA fragment containing the intervening intergenic spacer region (cpcBA-IGS) of 19 strains of three morphospecies of cyanobacteria. Six Australian strains were identified as Anabaena circinalis Rabenhorst, six strains were identified as Microcystis aeruginosa Kützing, and seven strains were identified as Nodularia spumigena Mertens. Restriction enzyme digestion of the amplification products from the strains revealed restriction fragment length polymorphism (RFLP) within all three morphospecies. Strains corresponding to M. aeruginosa were highly polymorphic: 11 of the 14 restriction enzymes used displayed RFLPs. The A. circinalis and N. spumigena strains were less variable: three of 14 enzymes and seven of 14 enzymes, respectively, showed RFLPs. The presence of genetic variation between strains within these three divergent morphospecies, which span two orders of cyanobacteria (Chroococcales Wettstein and Nostocales (Borzi) Geitler), show that the cpcBA- IGS fragment has broad application as a molecular marker for intrageneric studies of cyanobacteria systematics and genetics.     

Cloning and identification of the H-2D p gene

We have cloned six different class I genes from a B10.P sperm library. After cotransfection with the herpes simplex tk gene, one L-cell line was found to react with six H-2D^p-specific monoclonal antibodies. The cell line L12a did not react with K^p-specific monoclonal antibodies. This identification was confirmed by mapping a 2.5 kb Bam H 1 restriction fragment present in the 12a DNA clone to the D-TL region of H-2 ^p. Only a single 8.8 kb Barn H1 fragment can be assigned to K ^p by restriction fragment length polymorphism, while many others map to the D-TL interval. A restriction map of 12a is presented.     

Polymorphism and stability in the histone gene cluster of Drosophila melanogaster

Histone genes in Drosophila melanogaster are organized into repeats of 4.8 and 5.0 kb (Lifton et al., 1978). We find these repeat sizes in every one of the more than 20 Drosophila strains we have examined. Strains differ in the relative amounts of the two repeat types, with ratios varying from 11 to 14, the 5.0 kb repeat always present in equal or greater amounts than the 4.8 kb repeat. Restriction enzyme digestion and blotting analysis reveals that the strains also differ in a number of far less abundant fragments containing histone DNA sequences. In the Amherst and Samarkand strains, there are, in addition, many copies of 4.0 and 5.5 kb repeat-like fragments respectively. A series of stocks were made isogenic for single second chromosomes from the Amherst strain. The hybridization patterns of the histone DNA from these stocks containing different Amherst chromosomes are very similar but a number of differences in the minor fragments were seen. The stability of the histone locus restriction pattern was tested by following the DNA derived from a single second chromosome of the b Adhn² pr cn strain over a two year period. The restriction pattern of major and minor bands remained identical. Finally, histone loci distinguishable by their restriction pattern on blots were recombined with visible markers. These chromosomes will be useful in tracing the fate of specific histone loci during genetic manipulations.     

Characterization of relic DNA from barley genome

Summary High-molecular-weight relic DNA fraction can be electrophoretically separated from the bulk of barley DNA digested with different restriction enzymes. We have cloned and analyzed a population of relic DNA fragments. The majority of AluI-relic DNA clones contained barley simple sequence satellite DNA and other families of repetitive DNA. One of these families, designated HvRT, has been analyzed in detail. This family is composed of tandemly arranged 118-bp monomers and is present in 7 × 10⁵ copies in the barley genome. Clones representing the HvRT family were sequenced. HvRT repeats were found to contain high levels of methylated cytosine. The HvRT family was found in the genomes of H. vulgare, H. leporinum, H. murinum, H. jubatum, but not in H. marinum, H. geniculatum, and wheat. Different barley species and cultivars show restriction fragment length polymorphism with the HvRT probe. Chromosome-specific subfamilies of HvRT were found to be present on different barley chromosomes, providing the possibility of using the HvRT probe as a chromosome specific marker. HvRT fragments up to 810 kbp in length were resolved by pulsed field gel electrophoresis.     

The 5S ribosomal RNA gene clusters in Tetrahymena thermophila: strain differences, chromosomal localization, and loss during micronuclear ageing

Summary The organization of the 5S genes in the genome of Tetrahymena thermophila was examined in various strains, with germinal ageing, and the 5S gene clusters were mapped to the MIC chromosomes. When MIC or MAC DNA is cut with the restriction enzyme EcoRI, electrophoresed, blotted, and probed with a 5S rDNA probe, the banding patterns represent the clusters of the 5S rRNA genes as well as flanking regions. The use of long gels and 60 h of electrophoresis at 10 mA permitted resolution of some 30–35 5S gene clusters on fragments ranging in size from 30-2 kb (bottom of gel). The majority of the 5S gene clusters were found in both MIC and MAC genomes, a few being MIC limited and a few MAC limited. The relative copy number of 5S genes in each cluster was determined by integrating densitometric tracings made from autoradiograms. The total number of copies in the MAC was found to be 33% greater than in the MIC. When different inbred strains were examined, the majority of the 5S gene clusters were found to be conserved, with a few strain-specific clusters observed. Nine nullisomic strains missing both copies of one or more MIC chromosomes were used to map the 5S gene clusters. The clusters were distributed non-randomly to four of the five MIC chromosomes, with 17 of them localized to chromosome 1. A deletion map of chromosome 1 was constructed using various deletion strains. Some of these deletion strains included B strain clones which had been in continuous culture for 15 years. Losses of 5S gene clusters in these ageing MIC could be attributed to deletions of particular chromosomes. The chromosomal distribution of the 5S gene clusters in Tetrahymena is unlike that found for the well-studied eukaryotes, Drosophila and Xenopus.