The generation of a library of PCR-analyzed microsatellite variants for genetic mapping of the mouse genome

Forty-three sequences containing simple sequence repeats or microsatellites were generated from an M13 library of total genomic mouse DNA. These sequences were analyzed for size variation using the polymerase chain reaction and gel electrophoresis without the need for radiolabeling. Seventy-two percent of the sequences showed allelic size variations between different inbred strains of mouse and the wild mouse, Mus spretus; and 53% showed variation between inbred strains. Thirty-seven percent were variant between B6/J and DBA/2J, and 81% of these were resolved using minigel agarose electrophoresis alone. This approach is a useful way of generating the large number of variants that are needed to create high resolution maps of the mouse genome.     

PCR-analyzed microsatellites of the mouse genome—additional polymorphisms among ten inbred mouse strains

Eighty sequences from the mouse genome database containing microsatellites (simple sequence repeats) have been analyzed for size variation among ten different inbred strains of mice; 62/80 (77.5%) showed polymorphism of at least three alleles. We have been able to detect all the polymorphims by agarose gel electrophoresis, often running the gels for up to 3 h. Between individual pairs of mouse strains to be used in chromosomal mapping studies in our laboratory, 35–60% polymorphism occurred. There are potentially enough microsatellites within the mouse and human genome to have a marker at every 1-cM distance. This simple approach will, therefore, continue to be useful in genome mapping studies, leading eventually to high-resolution maps of both the mouse and human genomes; this should allow for physical mapping and cloning of specific genes.     

Most of the murine leukemia virus sequences in the DNA of NIH/swiss mice consist of two closely related proviruses, each repeated several times

The structure of the endogenous murine leukemia virus (MuLV) sequences of NIH/Swiss mice was analyzed by restriction endonuclease digestion, gel electrophoresis, and hybridization to an MuLV nucleic acid probe. Digestion of mouse DNA with certain restriction endonucleases revealed two classes of fragments. A large number of fragments (about 30) were present at a relatively low concentration, indicating that each derived from a sequence present once in the mouse genome. A smaller number of fragments (one to five) were present at a much higher concentration and must have resulted from sequences present multiple times in the mouse genome. These results indicated that the endogenous MuLV sequences represent a family of dispersed repetitive sequences. Hybridization of these same digested mouse DNAs to nucleic acid probes representing different portions of the MuLV genome allowed construction of a map of the sites where restriction endonucleases cleave the endogenous MuLV sequences. Several independent recombinant DNA clones of endogenous MuLV sequences have been isolated from C3H mice (Roblin et al., J. Virol. 43:113-126, 1982). Analysis of these sequences shows that they have the structure of MuLV proviruses. The sites at which restriction endonucleases cleave within these proviruses appeared to be similar or identical to the sites at which these nucleases cleaved within the MuLV sequences of NIH/Swiss mice. This identity was confirmed by parallel electrophoresis. We conclude that the apparently complex pattern of endogenous MuLV sequences of NIH/Swiss mice consists largely of only two kinds of provirus, each repeated multiple times at dispersed sites in the mouse genome.     

Coupling of polyoma DNA and RNA synthesis

Polyoma virus RNA from infected mouse embryo cells was examined by gel electrophoresis and in nucleic acid hybridisation experiments. The extent of representation of the polyoma genome in RNA sequences when cytosine arabinoside is present throughout infection is 30 to 40% of that at late times in infection. When viral DNA synthesis is inhibited during the period in which it is rising to its maximum, the resulting cytoplasmic RNA resembles ‘early’ RNA both in size and by its behaviour in competition hybridisation experiments.     

Size and physical map of the Campylobacter jejuni chromosome.

The chromosome of Campylobacter jejuni is circular and approximately 1700 kb in circumference. The size of the genome was determined by field inversion gel electrophoresis of restriction endonuclease fragments using lambda DNA concatamers and yeast chromosomes to calibrate the size of the fragments. In view of the low (32-35%) G + C content of the campylobacter genome, enzymes that recognizes GC-rich sequences were used. Of the enzymes tested BssHII (G/C(G)CGC), NciI (CC/CGCG) and SalI (G/TCGAC) appeared to be usable. Hybridization of labeled fragments with two or more fragments from digests with a different restriction enzyme gave the information to order the fragments on the C jejuni chromosome. The localization on the genome of the flagellin and ribosomal gene clusters was determined.     

Comparative analysis of genome sizes in Streptococcus thermophilus strains

Twenty-five Streptococcus thermophilus isolates were analyzed using pulse-field gel electrophoresis (PFGE) and gene restriction profile analysis techniques. 16S rRNA gene sequences of the isolates were almost 100% homologous. However, genomic fingerprinting analysis has shown variability in both genome size and restriction fragments length. The genomes varied from 1417 to 2075 kb resulting in the difference between marginal genome sizes in about 600 kb. The results are indicative of Streptococcus thermophilus intraspecies genetic polymorphism, the origin of which requires further investigation.     

Genome analysis of Pseudomonas aeruginosa by field inversion gel electrophoresis

Abstract The genome of Pseudomonas aeruginosa was analysed by digestion with rare-cutting restriction endonucleases and subsequent field inversion gel electrophoresis (FIGE). P. aeruginosa strain PAO and the 17 IATS strains were investigated. Each strain displayed a unique pattern of restriction fragments. Digestion with Dra I and Ssp I yielded, respectively 7–11 and 2–5 fragments of more than 130 kb in size, indicating the non-random occurrence of AT-rich sequences in the P. aeruginosa genome. The genome size of P. aeruginosa PAO was estimated to be (2.2 ± 0.3) × 10⁶ bp. The applications of DNA fingerprinting for gene cloning, construction of a physical chromosome map, and epidemiological studies, are discussed.     

A family of moderately repetitive sequences in mouse DNA.

When mouse DNA is digested to completion with restriction endonuclease Eco R1, a distinct band of 1.3 kb segments comprising about 0.5-3% of the genome is observed upon agarose gel electrophoresis. This DNA is not tandemly repeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kb band and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 10(4) times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco R1 band.     

Comparative analysis of genome sizes of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> strains

Twenty-five Streptococcus thermophilus isolates were analyzed using pulse-field gel electrophoresis (PFGE) and gene restriction profile analysis techniques. 16S rRNA gene sequences of the isolates were almost 100% homologous. However, genomic fingerprinting analysis has shown variability in both genome size and restriction fragments length. The genomes varied from 1417 to 2075 kb resulting in the difference between marginal genome sizes in about 600 kb. The results are indicative of Streptococcus thermophilus intraspecies genetic polymorphism, the origin of which requires further investigation.     

Temporal analysis of French Bordetella pertussis isolates by comparative whole-genome hybridization

Bordetella pertussis, a gram-negative beta-proteobacterium, is the agent of whooping cough in humans. Whooping cough remains a public health problem worldwide, despite well-implemented infant/child vaccination programs. It continues to be endemic and is observed cyclically in vaccinated populations. Classical molecular subtyping methods indicate that genome diversity among B. pertussis isolates is limited. Although the whole bacterial genome has been studied by pulsed-field gel electrophoresis, the genes implicated in the diversity have not been identified. We developed a B. pertussis whole-genome DNA microarray representing over 91% of the predicted coding sequences of the sequenced strain Tohama I. Genomic DNA from clinical isolates with various pulsed-field gel electrophoresis profile patterns was competitively hybridized with the DNA microarray and coding sequences were classified as present, absent or duplicated. Our data strongly suggest that the B. pertussis population is dynamic. In France, with highly vaccinated population, the genetic diversity is low and decreasing with time, and clonal expansion correlates with cycles of the disease. This decrease in diversity is essentially due to loss of genes and pseudogenes. The genes deleted are most of the time flanked by insertion sequences.     

Endogenous xenotropic murine leukemia virus-related sequences map to chromosomal regions encoding mouse lymphocyte antigens.

DNAs of all inbred mouse strains contain multiple copies (18 to 28 copies per haploid mouse genome) of endogenous xenotropic murine leukemia virus-related sequences detectable by Southern analysis with a xenotropic murine leukemia virus env gene-specific probe. After PvuII digestion, we identified a subset of xenotropic murine leukemia virus-related sequences that are well resolved by agarose gel electrophoresis and can be mapped to specific chromosomes by using recombinant inbred mouse strains. Interestingly, three of six xenotropic proviral loci that we mapped were integrated near genes encoding mouse lymphocyte antigens (Ly-m22, chromosome 1; Ly-m6, chromosome 2; and Ly-m10, chromosome 19) and a fourth xenotropic proviral locus mapped near a gene on chromosome 4 that has a major influence on xenotropic virus cell surface antigen levels. These studies indicate that xenotropic proviral loci are located on many different mouse chromosomes and may be useful markers for molecularly cloning and characterizing regions of the mouse genome important in lymphocyte development.     

On the mode of evolution of alpha satellite DNA in human populations

Summary The hypothesis that highly reiterated satellite DNAs in present-day populations evolve by molecular mechanisms that create, by saltatory amplification steps, new long arrays of satellite DNA, and that such long arrays are used for homogenization purposes, has been tested both in mouse and in humans. In mouse, the data obtained are consistent with this hypothesis. This was tested in more detail on chromosomes 13 and 21 of the human genome. A Centre d'Etudes du Polymorphisme Humain family, which in some individuals exhibits strong supplementary DNA bands following TaqI restriction endonuclease digestion and conventional gel electrophoresis, was analyzed by pulse field gel electrophoresis following restriction by BamHI. The supplementary bands on chromosome 13 (18 times the basic alpha satellite DNA repeat) and on chromosome 21 (a 9.5-mer) segregated with centromeric alpha satellite DNA blocks of 5 and 5.3 megabases, respectively. These are by far the largest alpha satellite block lengths seen in all chromosome 13 and chromosome 21 centrometric sequences so far analyzed in this manner. The possibility that these supplementary alpha satellite sequences were created in single individuals by saltatory amplification steps is discussed in light of our own data and that published by others. It is proposed that deletion events and unequal cross-overs, which both occur in large satellite DNA arrays, contribute to the homogenization of size and sequence of the alpha satellite DNA on most chromosomes of humans.     

Novel Mouse Microsatellites: Primer Sequences and Chromosomal Location

Sixty-nine sequences containing microsatellites were determinedby analysis of clones from a pUC118 library of total genomicmouse DNA. These sequences were examined for size variationusing polymerase chain reaction and gel electrophoresis. Fifty-oneof them showed allelic variations between C57BL/6 and MSM, thetwo strains used for genetic mapping. Hence, their chromosomallocation was determined using a panel consisting of 131 backcrossmice that had been typed with 85 anchor loci. The microsatelliteswere distributed to most chromosomes except for chromosomes16 and 19. These novel markers with defined locations are usefulin linkage and genome mapping studies.     

Screening of Rice Genes from the cDNA Catalog Using the Data Obtained by Protein Sequencing

The partial amino acid sequences of 121 rice proteins separated by two-dimensional gel electrophoresis (2D-PAGE), were determined for a protein sequence data file. In the Rice Genome Research Program (RGP), more than 20,000 cDNA clones randomly selected from rice cDNA libraries have been sequenced to construct a cDNA catalog. Complimentary DNAs encoding about 30% of proteins in the protein sequence data file could be identified in the catalog by computer search. It was deduced that 20,000–40,000 genes are present in the rice genome. Only half of about 20,000 cDNAs sequenced in the RGP, corresponding to 1/4–1/2 of genes present in the entire rice genome, should have unique sequences after considering gene redundancy. This is consistent with the fact that the cDNAs encoding about 30% of the sequenced proteins could be identified in the catalog. If the size of the cDNA catalog is enlarged further, cDNAs encoding all proteins separated by 2D-PAGE could be easily identified from the catalog by using the protein sequence data.     

Identification of mouse mammary tumor virus-specific mRNA.

Complementary DNA corresponding to the RNA genome of mouse mammary tumor virus was used to identify viral RNA contained in polysomes of a virus-producing mammary tumor cell line. Separation of polysomal mRNA by agarose gel electrophoresis, transfer of the RNA to diazobenzyloxymethyl paper, and hybridization with 32P-labeled mouse mammary tumor virus complementary DNA revealed three viral RNA size classes of 10, 8.8, and 4.4 kilobases in length, respectively.     

Size determination of poly(A) after in vitro methylation with radioactive dimethyl sulfate

A simple method for the size determination of poly(A) using in vitro labeling by methylation with [³H]dimethyl sulfate is described. After methylation, modified poly (A) has the same mobility, using polyacrylamide gel electrophoresis, as has the unmodified polymer, thus showing that the methylation does not cause degradation. Therefore the method is a sensitive assay to size the poly(A) segments from in vivo unlabeled tissue. The method was applied to determine the size of poly(A) sequences on mRNA from mouse L5178Y cells and from rat liver.     

Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite

Cytologically, the centromere is found at the very end of most Mus musculus chromosomes, co-localizing with an array of minor satellite sequences. It is separated from the euchromatin of the long arm by a large domain of heterochromatin, composed in part of arrays of major satellite sequences. We used oligonucleotide probes that specifically detect regions of sequence variation found in certain cloned minor satellite sequences. They detect a limited subset of the minor satellite arrays in the mouse genome, based on both pulsed-field gel electrophoresis and in situ hybridization data, and provide direct molecular genetic markers for individual centromeres in some inbred mouse strains. Array size polymorphisms detected by these probes map to positions consisten with the centromeres of chromosomes 1 and 14 in the BXD recombinant inbred (RI) strains. The genetic distances between these minor satellite arrays and loci on the long arms of chromosomes 1 and 14 are consistent with repression of meiotic recombination in the heterochromatic domains separating them. The existence of chromosome-specific minor satellite sequences implies that the rate of sequence exchange between non-homologous chromosomes relative to the rate between homologous chromosomes is much lower than has previously been postulated. We suggest that the high degree of sequence homogeneity of mouse satellite sequences may instead reflect recent common ancestry.