Insertion-sequence-dependent rearrangements of Pseudomonas cepacia plasmid pTGL1.

Pseudomonas cepacia 249 (ATCC 17616) harbors a 170-kilobase (kb) plasmid designated pTGL1. We identified three insertion sequences, IS405, IS408, and IS411, on this plasmid. Various prototrophic and auxotrophic derivatives in our collection contained variants of pTGL1 formed by accretion and deletion of other elements. Plasmid pTGL6, the variant in one prototroph, evolved from pTGL1 by the addition of three copies of IS401 (1.3 kb) and one of IS402 (1 kb), to generate pTGL5, and recombination between two of the copies of IS401 on pTGL5 to form pTGL6. The latter event entailed loss of one copy of IS401 and an additional 5.4 kb of plasmid DNA. Derivatives of the broad-host-range plasmid pRP1 carrying the above insertion sequences and recombinant plasmids carrying fragments of plasmids pTGL6 and pTGL5 were used as probes to ascertain the extent of reiteration of the various elements in the P. cepacia genome. The data indicate a high frequency of genomic rearrangements which presumably contributes to the extraordinary adaptability of this bacterium.     

The molecular basis of I-R hybrid dysgenesis in Drosophila melanogaster: identification, cloning, and properties of the I factor

We have analyzed two mutations of the white-eye gene, which arose in flies subject to I-R hybrid dysgenesis. These mutations are associated with insertions of apparently identical 5.4 kb sequences, which we have cloned. We believe that these insertions are copies of the I factor controlling I-R hybrid dysgenesis. The I factor is not a member of the copia-like or fold-back classes of transposable elements and has no sequence homology with the P factor that controls P-M dysgenesis. All strains of D. melanogaster contain I-factor sequences. Those present in reactive strains must represent inactive I elements. I elements have a remarkably similar sequence organization in all reactive strains and are located in peri-centromeric regions. Inducer strains appear to contain both I elements, located in peri-centromeric regions, and 10-15 copies of the complete I factor at sites on the chromosome arms.     

A sensitive assay for detecting mutations resulting from unequal homologous recombination without phenotypic selection

Naturally occurring mutations are composed of a large number of mutations of many types that include mutations resulting from unequal homologus recombination between repetitive elements. The present paper describes a sensitive method for detecting such mutations without phenotypic selection. This system utilizes a tandemly arranged Vr repetitive sequence comprising 6000 copies in the mouse genome that is present in the spacer of the ribosomal RNA gene. For HincII digests of FM3A cell DNA, the Vr probe provides 4 major bands of 2.7 kb, 1.7 kb, 1.6 kb and 1.35 kb and several minor bands. Newly induced mutations due to unequal homologous recombination are observed as disappearance of the minor bands and appearance of extra bands. With this method a spontaneous mutation was detected in 14 cell clones randomly isolated after 60 days of continuous growth. Exposure to 4 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine for 2 h revealed 4 mutations in 11 clones examined. Culturing the cells after treatment in the presence of 12-O-tetradecanoylphorbol 13-acetate enhanced the frequency, yielding 6 mutations in 5 clones. The assay can skip phenotypic selection prior to analysis of DNA changes and hence provides a direct method for monitoring mutations resulting from homologous recombination in non-biased cell populations.     

Physical mapping and cloning of a translocation in sugar beet (Beta vulgaris L) carrying a gene for nematode (Heterodera schachtii) resistance from B. procumbens

Two diploid (2n=18) sugar beet (Beta vulgaris L.) lines which carry monogenic traits for nematode (Heterodera schachtii Schm.) resistance located on translocations from the wild beet species Beta procumbens were investigated. Short interspersed repetitive DNA elements exclusively hybridizing with wild beet DNA were found to be dispersed around the translocations. The banding pattern as revealed by genomic Southern hybridization was highly conserved among translocation lines of different origins indicating that the translocations are not affected by recombination events with sugar beet chromosomes. Physical mapping revealed that the entire translocation is represented by a single Sal I fragment 300 kb in size. A representative YAC (yeast artifical chromosome) library consisting of approximately 13,000 recombinant clones (2.2 genome equivalents) with insert sizes ranging between 50 and 450 kb and an average of 130kb has been constructed from the resistant line A906001. Three recombinant YACs were isolated from this library using the wild beet-specific repetitive elements as probes for screening. Colinearity between YAC inserts and donor DNA was confirmed by DNA fingerprinting utilizing these repetitive probes. The YACs were arranged into two contigs with a total size of 215 kb; these represent a minimum of 72% of the translocation.     

DNA profiling in a genetically isolated population using three hypervariable DNA markers.

We determined the allele frequencies for three hypervariable DNA loci D2S44, D1S7 and D7S21 using the probes YNH24, MS1 and MS31 in the genetically isolated Finnish population. The allelic length ranges were 1.7- < 6.0 kb for YNH24, 1.7- < 18.0 kb for MS1 and 3.2- < 12.0 kb for MS31. High heterozygosity rates (0.94-0.96) were detected for all three probes. In 48 mother-child pairs no mutations were found using the probes YNH24 and MS31, whereas a mutation rate of 0.064 was observed for probe MS1. In addition, an unexpected four-band pattern was detected in 1 out of 170 individuals using the probe MS1, suggesting complex DNA polymorphism based on both a variable number of tandem repeats and restriction site polymorphism. Our findings also show that all three probes are valuable in individual identification in this genetically isolated population.     

Characterization of common BRCA1 and BRCA2 variants

Many missense variants identified in BRCA1 and BRCA2, two genes responsible for the majority of hereditary breast and ovarian cancer, are of unclear clinical significance. Characterizing the significance of such variants is important for medical management of patients in whom they are identified. The aim of this study was to characterize eight of the most common reported missense mutations in BRCA1 and BRCA2 occurring in patients tested for hereditary risk of breast and ovarian cancers. The prevalence of each variant in a control population, co-segregation of the variant with cancer within families, location of the variant within the gene, the nature of the amino acid substitution and conservation of the wild-type amino acid among species were considered. In a control population, the BRCA1 variants M1652I, R1347G, and S1512I, were each observed at a frequency of 4.08%, 2.04%, and 2.04%, respectively, and the BRCA2 variants A2951T, V2728I, and D1420Y, were seen at 1.02%, 0.68%, and 0.34%, respectively. Although the BRCA2 variants T598A and R2034C were not seen in this group of controls, other clinical and published observations indicate that these variants are not deleterious. Based on epidemiological and biological criteria, we therefore conclude that the BRCA1 missense mutations R1347G, S1512I and M1652I, and the BRCA2 missense mutations T598A, D1420Y, R2034C, V2728I, and A2951T, are not deleterious mutations.     

Systematic identification of LINE-1 repetitive DNA sequence differences having species specificity between Mus spretus and Mus domesticus

LINE-1 is a family of repetitive DNA sequences interspersed among mammalian genes. In the mouse haploid genome there are about 100,000 LINE-1 copies. We asked if the subspecies Mus spretus and Mus domesticus have developed species-specific LINE-1 subfamilies. Sequences from 14 M. spretus LINE-1 elements were obtained and compared to M. domesticus LINE-1 sequences. Using a molecular phylogenetic tree we identified several differences shared among a subset of young repeats in one or the other species as candidates for species-specific LINE-1 variants. Species specificity was tested using oligonucleotide probes complementary to each putative species-specific variant. When hybridized to genomic DNAs, single-variant probes detected an expanded number of elements in the expected mouse. In the other species these probes detected a smaller number of matches consistent with the average rate of random divergence among LINE-1 elements. It was further found that the combination of two species-specific sequence differences in the same probe reduced the detection background in the wrong species below our detection limit.     

A DNA segment from D. melanogaster which contains five tandemly repeating units homologous to the major rDNA insertion

We describe a cloned segment of D. melanogaster DNA (cDm219) that contains five tandemly arranged sequence units homologous to the type I insertion sequence found in the majority of 28S rRNA genes on the X chromosome. Heteroduplex studies show that two of the units have a deletion corresponding to a 1.1 kb piece of DNA close to the right-hand end of the type I insertion. Another unit has a 7.5 kb sequence (zeta) substituted for a 0.95 kb piece of DNA close to the left-hand part of the type I rDNA insertion. The two remaining units are interrupted by the Col E1 plasmid vector. There are also differences in the restriction endonuclease cleavage maps both between the units of cDm219 themselves and compared to the restriction endonuclease cleavage maps of cloned rDNA segments that contain type I insertions. Quantitation of the gel transfer hybridization of zeta element probes to restriction endonuclease digests of D. melanogaster DNA indicates there are 30--40 copies of zeta sequences distributed in seven major arrangements within the haploid genome. The hybridization of zeta and insertion sequence probes to a library of D. melanogaster DNA segments cloned in bacteriophage lambda indicates at least 4--6 copies of the zeta element could be linked to insertion sequences. The common site of in situ hybridization of zeta sequences is to the chromocentral heterochromatin of polytene chromosomes.     

An abundant family of methylated repetitive sequences dominates the genome of Physarum polycephalum.

A significant portion (20%) of the Physarum genome can be isolated as a HpaII-resistant, methylated fraction. Cloned DNA probes containing highly-repeated sequences derived from this fraction were used to define the pattern of structural organisation of homologous repeats in Physarum genomic DNA. It is shown that the probes detect an abundant, methylated family of sequences with an estimated genomic repetition frequency greater than 2100, derived from a large repeated element whose length exceeds 5.8kb. Sequences comprising the long repetitive element dominate the HpaII-resistant compartment and account for between 4-20% of the Physarum genome. Detailed restriction/hybridisation analysis of cloned DNA segments derived from this compartment shows that HpaII/MspI restriction sites within some copies of the long repeated sequence are probably deleted by mutation. Additionally, segments of the repeat are often found in different organisational patterns that represent scrambled versions of its basic structure, and which are presumed to have arisen as a result of recombinational rearrangement in situ in the Physarum genome. Preliminary experiments indicate that the sequences are transcribed and that the structural properties of the repeat bear some resemblance to those of transposable genetic elements defined in other eukaryotic species.     

The clustered and scrambled arrangement of moderately repetitive elements in Drosophila DNA.

An examination of cloned Drosophila DNA has revealed large clusters of densely spaced, short (less than or equal to 1 kb), moderately repetitive elements. Different clusters have many of the same repetitive elements, but these elements are arranged differently in each cluster. It is improbable that this clustered arrangement can be detected by conventional reassociation kinetic and electron microscopic techniques, but it can be detected and features of its fine structure can be determined by a two-dimensional version of Southern's blotting technique. The genomic organization of these clustered repetitive elements was investigated by hybridizing restriction fragments of cloned DNA to polytene chromosomes, to filter-bound recombinant DNA clones and to Southern blots of total Drosophila DNA. These studies demonstrated that clusters occur in euchromatic regions of the chromosomes and that at least one of the clusters has the same repetitive element organization in cloned and in chromosomal DNA. These studies also demonstrated that copies of the elements from one cluster are scattered in at least 1000 chromosomal regions. These regions appear to have differing concentrations of repetitive DNA, but together they account for a large fraction of Drosophila's moderately repetitive DNA. Aside from indicating the genomic organization of cluster elements, this work has identified cluster elements throughout a 9 kb region neighboring one of the heat shock genes, throughout the intron of the major rDNA repeat and within the apparently transposable element, 412.     

Novel detection of restriction fragment length polymorphisms in the human major histocompatibility complex

Cosmid genomic DNA clones have been used as hybridization probes in genomic Southern blot analysis to define restriction fragment length polymorphisms (RFLPs) in the major histocompatibility complex (MHC). Using 14 different enzymes and three overlapping cosmid clones we have detected six RFLPs in a 100 kilobase (kb) segment of DNA in the class III region extending centromeric of theTNFA gene towardHLA-DR. Four of the five RFLPs, defined using the enzymesTaqI,Rsa I,Hinc II, andHind III, and detected by the cosmid clone cosM7B, map to a 29 kb segment of DNA that includes all of the recently described G2 (BAT2) gene and a large portion of the 3 end of the G3 (BAT3) gene. The different RFLP variants were established by analyzing the DNA from three informative families and a panel of 51HLA-homozygous typing cell lines. CosM7B detectsTaq I variants of 4.3 kb, and 2.9 kb or 2.8 kb, Rsa I variants of 2.9 kb or 2.4 kb,Hinc II variants of 5.8 kb or 3.8 kb and 1.4 kb, and aHind III variant of 4.8 kb, while cosOT2 detects Taq I variants of 4.5 kb or 4 kb. The distribution of theRsa 1, Hinc II and Taq I RFLPs detected by cosM7B, and theTaq I RFLP detected with cosOT2, within the panel of cell line DNAs was assessed by Southern blotting. The 4.3 kbTaq I variant was observed in only one cell line with the extended haplotypeHLA-A29, C-, B44, SC30, DR4. The other RFLPs, however, occurred much more frequently. The 2.8 kb Taq I variant was observed in 20 % of haplotypes, the 2.9 kbRsa I variant was observed in 42% of haplotypes, and the 5.8 kbHinc I variant was observed in 12 % of haplotypes analyzed. The 4.5 kbTaq I variant detected by the overlapping cosmid cosOT2 was present in 21 % of haplotypes. Analysis of the RFLP variants with each other revealed seven different haplotypic combinations. Three of the haplotypic combinations were each subdivided into two subsets on the basis of the Nco I RFLP variant they carried at theTNF-B locus. These haplotypic combinations potentially allow differentiation among different extended haplotypes such asHLA-B8, SC01, DR3, HLA-B18, F1 C30, DR3, andHLA-B44, FC31, DR7. The RFLPs detected by the cosmid clones thus provide new tools which will be useful in the further genetic analysis of the MHC class III region.     

Isolation of BamHI variants with reduced cleavage activities

Derivation of the bamhIR sequence (Brooks, J. E., Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Ives, C. C., Mazzola, L. M., Slatko, B. E., and Benner, J. S. (1991) Nucleic Acids Res., in press), the gene coding for BamHI endonuclease, has facilitated construction of an Escherichia coli strain that overproduces BamHI endonuclease (W. E. Jack, L. Greenough, L. F. Dorner, S. Y. Xu, T. Strezelecka, A. K. Aggarwal, and I. Schildkraut, submitted for publication). As expected, low-level constitutive expression of the bamhIR gene in E. coli from the Ptac promotor construct is lethal to the host unless the bamHIM gene, which encodes the BamHI methylase, is also expressed within the cell. We identified four classes of BamHI endonuclease variants deficient in catalysis by selecting for survival of a host deficient for bamHIM gene, transformed with mutagenized copies of the bamhIR gene, and then screening the surviving cell extracts for DNA cleavage and binding activities. Class I variants (G56S, G91S/T153I, T114I, G130R, E135K, T153I, T157I, G194D) displayed 0.1-1% of the wild-type cleavage activity; class II variant (D94N) lacked cleavage activity but retained wild-type DNA binding specificity; class III variants (E77K, E113K) lacked cleavage activity but bound DNA more tightly; class IV variants (G56D, G90D, G91S, R122H, R155H) lacked both binding and cleavage activities. Variants with residual cleavage activities induced the E. coli SOS response and thus are presumed to cleave chromosomal DNA in vivo. We conclude that Glu77, Asp94, and Glu113 residues are essential for BamHI catalytic function.     

New tandem repeat region in the non-transcribed spacer of human ribosomal RNA gene.

A new repetitive DNA region was identified in the non-transcribed spacer of human rDNA, namely a long (4.6 kb) sequence motif (Xbal element) was present in two copies. The repeating unit composed of two parts. One of them consisted of unique nucleotide sequences, interrupted by some simple sequences. The other, about 3.1 kb long one assembled only from highly repeated simple sequences. The unique sequence region contained two, inverted copies of the human AluI type repetitive DNA family. The authors suggest that the XbaI elements may flank the tandem arrays of human rRNA genes as terminal repeats and they might function both as the origin of rDNA replication and/or site of homologous recombination.     

Structure and genomic organization of proretrovirus-like elements partially eliminated from the somatic genome of Ascaris lumbricoides.

A clone containing a middle repetitive element next to satellite DNA has been isolated from a germ line genomic library of the chromatin eliminating nematode Ascaris lumbricoides var. suum. The structure of this element has been elucidated by comparison of several clones containing the element in different environments. It is flanked by 256-bp-long terminal repeats (LTRs) and has an internal region of approximately 7 kb. The nucleotide sequences of both the 5' and the 3' LTRs have been determined. The element has a strong structural similarity with retroviral proviruses and related mobile elements. It was therefore named 'Tas', for transposon-like element of Ascaris. Approximately 50 Tas copies are dispersed over approximately 20 different chromosomal sites. Their genomic distribution varies between individuals, indicating that Tas elements are mobile in the Ascaris genome. Two variant forms, Tas-1 and Tas-2, present in a ratio of approximately 2 to 1 in the germ line genome, have been characterized. They differ not only in their restriction pattern, but also in their elimination behaviour. While only about one-fourth of the Tas-1 elements are expelled from the somatic cell lineage, all Tas-2 copies are specifically eliminated and are thus confined to the germ line cells. We have demonstrated that a cloned representative of Tas-1 elements is expelled concomitantly with its flanking DNA sequences during the chromatin elimination process.     

Recombination in simian virus 40-infected cells. Structure of naturally arising variants ev-2114, ev-2102, and ev-1110

The complete nucleotide sequence has been determined for three newly cloned evolutionary variants from two different independently generated evolutionary series (1100 and 2100 series) of simian virus 40 (SV40). These naturally arising variants, designated ev-1110, ev-2102, and ev-2114, were isolated after five high multiplicity serial passages. The structure of the variants consists of a monomeric unit tandemly repeated four times (ev-2102 and ev-2114) or six times (ev-1110) in the variant genome; the variants have four or six copies, respectively, of the viral origin signal for DNA replication. The DNA content in the three variants is vastly different in that the genome of variant ev-2114 contains only rearranged viral sequences, while variant ev-2102 contains a substitution with monkey DNA sequences consisting of a nearly complete dimeric unit of Alu family sequences as well as less repetitive sequences and variant ev-1110 contains monkey DNA sequences derived solely from repetitive alpha-component DNA. Recombination events, cellular sequences, and structural features of these and other naturally arising SV40 variants are compared.     

Mobile elements and transposition events in the cut locus of Drosophila melanogaster

We have cloned from the Oregon R strain of Drosophila melanogaster a 240 kb segment of DNA that contains the cut (ct) locus, and characterized the region for the presence of repetitive elements. Within this region at least five copies of the suffix element were detected, as well as several putatively novel mobile elements. A number of mutations obtained from the unstable ct ^MR2 strain and its derivatives were mapped within the cut locus. Comparison between parental and daughter strains indicates that frequently two or more independent transposition events involving the cut locus occur simultaneously within a single germ cell, thus providing a molecular basis for the transposition explosion phenomenon.     

Repetitive elements in the major histocompatibility complex (MHC) class I region of a teleost, medaka: identification of novel transposable elements

The repetitive elements of medaka (Oryzias latipes) are poorly characterized in spite of recent rapid progress in the medaka genome analysis. Here we report the characterization of the repetitive elements in the major histocompatibility complex (MHC) class I region, which spans about 400 kb and is one of the best characterized regions of the medaka genome. Microsatellite, low complexity regions, transposable elements, and other repeats occupied 0.68, 0.98, 7.0 and 2.9%, respectively, of the MHC class I region. Eleven transposable elements, three LTR-type, six LINE-type and two DNA-type, including several novel ones, were identified. Genomic Southern hybridization analysis indicated that these LINE-type and DNA-type elements have many copies in the medaka genome, whereas the LTR-type elements have only several copies. The comparison of the medaka MHC class I region with those of zebrafish and fugu shows the presence of three medaka lineage-specific tandem duplications of the PSMB (proteasome beta-type subunit) 8 and class Ia genes. Since eight of the 11 transposable elements were located in this region, these elements may have played a role in the medaka-specific DNA rearrangement.     

Copy number increases of transposable elements and protein‐coding genes in an invasive fish of hybrid origin

Evolutionary dynamics of structural genetic variation in lineages of hybrid origin is not well explored, although structural mutations may increase in controlled hybrid crosses. We therefore tested whether structural variants accumulate in a fish of recent hybrid origin, invasive Cottus, relative to both parental species Cottus rhenanus and Cottus perifretum. Copy‐number variation in exons of 10,979 genes was assessed using comparative genome hybridization arrays. Twelve genes showed significantly higher copy numbers in invasive Cottus compared to both parents. This coincided with increased expression for three genes related to vision, detoxification and muscle development, suggesting possible gene dosage effects. Copy number increases of putative transposons were assessed by comparative mapping of genomic DNA reads against a de novo assembly of 1,005 repetitive elements. In contrast to exons, copy number increases of repetitive elements were common (20.7%) in invasive Cottus, whereas decrease was very rare (0.01%). Among the increased repetitive elements, 53.8% occurred at higher numbers in C. perifretum compared to C. rhenanus, while only 1.4% were more abundant in C. rhenanus. This implies a biased mutational process that amplifies genetic material from one ancestor. To assess the frequency of de novo mutations through hybridization, we screened 64 laboratory‐bred F₂ offspring between the parental species for copy‐number changes at five candidate loci. We found no evidence for new structural variants, indicating that they are too rare to be detected given our sampling scheme. Instead, they must have accumulated over more generations than we observed in a controlled cross.     

Selective expansion of the newly evolved genomic variants of retrotransposon 1731 in the Drosophila genomes

The structural variants of the regulatory and coding regions of the LTR-retrotransposon 1731 are described. Two classes of genomic copies of retrotransposon 1731, with and without frameshifting strategy to express Gag and Pol proteins, were earlier revealed in the D. melanogaster genome. Copies without frameshifting are shown to be evolved from an ancient variant with frameshifting and are widespread in the genomes of the melanogaster complex species. Position of a rare codon responsible for ribosome pausing and efficient frameshifting is identified. Two structural variants of 1731 LTRs were detected in the melanogaster complex species: the predominant structural variant A1A2 of 1731 LTR in the D. melanogaster, D. simulans, and D. sechellia genomes contains duplicated and diverged copies of 28 bp in the U3 region, whereas A1 variant lacking this duplication is expanded in the D. mauritiana genome. Selective expansion of the A1A2 variant was detected in the independently established D. melanogaster cell cultures. A1A2 variant is expressed in embryos, cell culture, and testes, whereas A1 is expressed only in testes of D. melanogaster. Relief of expression of the A1A2 but not A1 variant in the ovaries as a result of mutation in the RNA interference (RNAi) spn-E gene is shown. Thus, expansion of the recently evolved genomic variants of the LTR retrotransposon 1731 possessing a new translation strategy, duplication in the U3 region, and extended profile of expression is revealed.