Sequence rearrangement in the AT-rich minisatellite of the novel rice transposable element Basho.

In the process of characterizing a rice wx deletion mutant, an AT-rich minisatellite sequence that consisted of units of approximately 80 bp was detected about 2.3 kb downstream of the wx gene. This AT-rich minisatellite was a multiple-copy element (1 x 10(3) to 2 x 10(3) copies per haploid genome) and interspersed in the rice genome. By BLAST homology search it was indicated that not only the tandem repeat but also both flanking sequences were conserved among copies. According to the characteristics of the termini (5'-CHH ... CTAG-3') and a target site preference for T, this AT-rich minisatellite accompanying the flanking sequences was classified into a novel transposon, Basho. The results of direct amplification of Basho showed that relatively large variation in size existed in the Basho family. We estimate the variation to be generated by not only alteration of the number of units in the minisatellite but also by duplications of larger blocks including the conserved flanking sequences caused by single-strand mispairing (SSM) at noncontiguous repeats. Because the AT-rich minisatellite contained in Basho possessed several motifs of the matrix attachment region (MAR) in its repeat unit, the functional role as MAR in the rice genome was discussed.     

Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46

Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into lambda gt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2.3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley beta-glucan, Avicel, filter paper and p-nitrophenyl beta-D-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48,863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.     

Evolutionary conservation of the apolipoprotein E-C1-C2 gene cluster on bovine chromosome 18q24

We have constructed a long-range restriction map spanning about 250 kb on bovine chromosome 18q24. Our results show that the apolipoprotein C2 (APOC2) gene is located about 25 kb from the APOE gene. Four putative CpG islands are also indicated in the map. Interestingly, a minisatellite located in the third intron of the human and mouse APOC2 genes was also found at identical position in the bovine gene and revealed high sequence identity comparing with the two corresponding sequences. By means of cosmid mapping, we further demonstrate that the APOE-APOC1-APOC2 gene cluster is evolutionary conserved in cattle.     

Genetic variance of Trichomonas vaginalis isolates by Southern hybridization

In the present study, genomic DNAs were purified from Korean isolates (KT8, KT6, KT-Kim and KT-Lee) and foreign strains (CDC85, IR78 and NYH 286) of Trichomonas vaginalis, and hybridized with a probe based on the repetitive sequence cloned from T. vaginalis to observe the genetic differences. By Southern hybridization, all isolates of T. vaginalis except the NYH286 strain had 11 bands. Therefore all isolates examined were distinguishable into 3 groups according to their banding patterns; i) KT8, KT6 and KT-Kim isolates had 11 identical bands such as 1 kb, 1.2 kb, 1.6 kb, 1.9 kb, 2.3 kb, 2.7 kb, 3.2 kb, 3.4 kb, 3.8 kb, 4.9 kb and 6.0 kb. ii) The metronidazole-resistant IR78 strain had the same bands as KT-Lee isolate at bands of 1 kb, 1.2 kb, 1.6 kb, 1.8 kb, 2.1 kb, 2.5 kb, 2.7 kb, 2.9 kb, 3.4 kb, 5.0 kb and 6.0 kb. Bands of CDC85, metronidazole-resistant strain, were similar to those of IR78 and KT-Lee, except that 3.2 kb replaced 2.9 kb. iii) NYH286 particularly had 12 bands and band patterns were similar to IR78 with a few exceptions as follows: i) 6.2 kb in place of 6.0 kb, ii) 2.0 kb and 2.2 kb instead of 2.1 kb. Through the results obtained, genetic variance of T. vaginalis isolates was demonstrated by Southern hybridization.     

A study for minisatellitic markers of Conidiobolus coronatus' pathogenicity to Galleria mellonella larvae

A selected panel of 13 colonies of entomopathogenic fungus Conidiobolus coronatus representing 6 variants of pathogenicity to Galleria mellonella larvae (ranged from 100 to 10% of efficiency), derived from single spores, were tested for the presence of hypervariable loci in their genomes by hybridization with Jeffreys' human minisatellite probe 33.6. The probe produced informative fingerprints and revealed slight differences among colonies analyzed. Up to 20 variable bands per colony were recognized in the size range of 2-20 kb. The band sharing within groups with the same pathogenicity ranged from 0.966 to 0.800. The genetic distance between different variants ranged from 0.026 to 0.282. A few characteristic bands for high and low pathogenicity to the larvae were found.     

Construction of genomic libraries of Cryptosporidium parvum and identification of antigen-encoding genes.

Genomic libraries have been constructed from bovine C. parvum DNA in the lambda ZAP and lambda DASH vectors. Based on an estimated genome size of 2 x 10(4) kilobases (kb), each recombinant library contains greater than 10 genomic equivalents. The average recombinant size for the lambda ZAP library is 2.1 kb and for the lambda DASH library is 14 kb. We have identified genes to major antigens recognized by hyperimmune bovine antiserum. These recombinants are currently being purified and characterized. Limited DNA sequence analysis of random C. parvum clones confirms suggestions that the genome is quite AT-rich. The DNA sequence of random lambda ZAP fusion proteins has identified a potential ATPase, a structural protein and a DNA-binding protein.     

Molecular evolution of a plastid tandem repeat locus in an orchid lineage

The molecular evolution of a chloroplast minisatellite locus in the Anacamptis palustris (Orchidaceae) lineage and haplotype variation in two Italian A. palustris populations were investigated. A phylogenetic analyses of the chloroplast tRNA(LEU) intron, where the minisatellite locus is located, revealed that a deletion in the ancestor of the A. palustris lineage led to the formation of two noncontiguous, complementary sequence motifs. We propose a model to explain the initial formation of the minisatellite repeat motif, starting with the two noncontiguous, complementary sequence motifs. A survey of minisatellite variation in four species of the A. palustris lineage revealed several haplotypes that differed not only in repeat number, but also in repeat organization. A haplotype network suggests that three different minisatellite loci evolved independently at the same position in the tRNA(LEU) intron. A secondary structure model revealed that the A. palustris minisatellite repeat forms a stem region of the tRNA(LEU) intron, which allows its notable expansion without negatively affecting splicing. Minisatellite variation was high in the two examined A. palustris populations where 20 haplotypes were detected, whereas no length variation was detected in a neighboring poly (A) microsatellite locus. We estimated a chloroplast minisatellite mutation rate of 3.2 x 10(-3) mutations per generation. Southern blot analyses did not find evidence for chloroplast heteroplasmy. Based on the analysis of the largest known, extant A. palustris population, a stepwise mutation model (SMM) was inferred.     

Characterization of a human alphoid satellite DNA sequence: Potential use in assessing genetic diversity in Indian populations

Sequence analysis of a human repetitive DNA sequence (pTRF5.6) revealed considerable homology (76%) to the alphoid consensus sequence. Genomic blots of StuI-digested human DNA, hybridized to pTRF5.6, generated a ladder of bands with each band corresponding to oligodeoxyribonucleotide of an approx. 170-bp repeat, indicating a tandemly arrayed organization of this repeat element within the genome. Genomic hybridization analyses of unrelated individuals belonging to various geographical regions of India, using this alphoid satellite probe, revealed polymorphic bands ranging between 2 and 9 kb. Along with an individual-specific band pattern, several isomorphic bands below 2 kb were also evident. There was very little of genetic variability between populations, suggestive of low polymorphism at the inter-population level. Our result suggest that alphoid satellite sequence probe can be used in assessing the genetic diversity of various ethnic groups/populations belonging to different geographical regions.     

The human minisatellite consensus at breakpoints of oncogene translocations.

A reexamination of human minisatellite (hypervariable) regions following the cloning and sequencing of the new minisatellite, VTR1.1, revealed that many of these structures possessed a strongly conserved copy of the chi-like octamer, GC[A/T]GG[A/T]GG. In oncogene translocations apparently created by aberrant VDJ recombinase activity, this VTR octamer was often found within a few bases of the breakpoint (p less than 10(-10)). Three bcl2 rearrangements which occurred within 2 bp of one another were located precisely adjacent to this consensus; it defined the 5' border of that oncogene's major breakpoint cluster. Several c-myc translocations also occurred within 2 bp of this sequence. While the appearance of a chi-like element in polymorphic minisatellite sequences is consistent with a role promoting either recombination or replication slippage, the existence of such elements at sites of somatic translocations suggests chi function in site-specific recombination, perhaps as a subsidiary recognition signal in immunoglobulin gene rearrangement. We discuss the implications of these observations for mechanisms by which oncogene translocations and minisatellite sequences are generated.