Could n-gram analysis contribute to genomic island determination?

There are two approaches to identifying genomic and pathogenesis islands (GI/PAIs) in bacterial genomes: the compositional and the functional, based on DNA or protein level composition and gene function, respectively. We applied n-gram analysis in addition to other compositional features, combined them by union and intersection and defined two measures for evaluating the results—recall and precision. Using the best criteria (by training on the Escherichia coli O157:H7 EDL933 genome), we predicted GIs for 14 Enterobacteriaceae family members and for 21 randomly selected bacterial genomes. These predictions were compared with results obtained from HGT DB (based on the compositional approach) and PAI DB (based on the combined approach). The results obtained show that intersecting n-grams with other compositional features improves relative precision by up to 10% in case of HGT DB and up to 60% in case of PAI DB. In addition, it was demonstrated that the union of all compositional features results in maximum recall (up to 37%). Thus, the application of n-gram analysis alongside existing or newly developed methods may improve the prediction of GI/PAIs.     

Integration of vectors by homologous recombination in the plant pathogen Glomerella cingulata

An homologous transformation system has been developed for the plant pathogenic fungus Glomerella cingulata (Colletotrichum gloeosporioides). A transformation vector containing the G. cingulata gpdA [romoter fused to the hygromycin phosphotransferase gene was constructed. Southern analyses indicated that this vector integrated at single sites in most transformants. A novel method of PCR amplification across the recombination junction point indicated that the integration event occurred by homologous recombination in more than 95% of the transformants. Deletion studies demonstrated that 505 bp (the minimum length of homologous promoter DNA analysed which was still capable of promoter function) was sufficient to target integration events. Homologous integration of the vector resulted in duplication of the gdpA promoter region. When transformants were grown without selective pressure, a high incidence of vector excision by recombination between the duplicated regions was evident. The significance of these recombination characteristics is discussed with reference to the feasibility of performing gene disruption experiments.     

Genome diversity in temperate bacteriophages of Oenococcus oeni

Summary.  The genome structure of six bacteriophages of Oenococcus oeni was compared. Two distinct groups with no apparent restriction site conservation were defined. In members of the α group (fOgML34, fOg4029, fOg30 and fOg218) a 7.5 kb region containing the origin of DNA packaging (cos) was highly conserved. Stretches of DNA heterogeneity could also be assigned to particular regions and were mostly evident in the right area of the genomes. fOg44 and fOgPSU1 (β group) were indistinguishable in the left half of their genomes, including cos, but were markedly dissimilar in other regions. Strong labelling signals detected in cross-hybridizations involving members of different groups were confined to fragments centrally located in their physical maps. The attachment site (attP) of fOg44 was assigned to this conserved region. It is suggested that recombination events at this location may have been important in generating the observed diversity of oenophage genomes. Accepted October 15, 1997 Received August 11, 1997     

The most frequent DCLRE1C (ARTEMIS) mutations are based on homologous recombination events

The nuclease ARTEMIS is an essential factor of V(D)J recombination during lymphocyte development and in the repair of DNA double‐strand breaks (DSB) by the nonhomologous end joining (NHEJ) pathway. Patients with mutations in the DCLRE1C gene, which encodes ARTEMIS, suffer from radiosensitive B^?/low T^?/low severe combined immunodeficiency (SCID) or radiosensitive Omenn syndrome. To date, causative DCLRE1C mutations inherited as a recessive trait have been reported in 49 patients. In this study, molecular diagnoses of 29 novel patients presenting with the phenotype of B^?/low SCID revealed mutations in the DCLRE1C gene. In total, 13 different mutated DCLRE1C alleles were detected, nine of which have not been described before. By far the most frequent mutations (59%) were gross deletions of exons 1–3 or exons 1–4 due to a homologous recombination of the wild‐type DCLRE1C gene with a pseudo‐DCLRE1C gene located 61.2 kb 5′ to the DCLRE1C start codon. Fine mapping of the recombination intervals revealed private mutations in most cases. MEIG1, a gene encoding a protein that is essential for spermatogenesis in mice, is lost by the gross deletions. Functional analyses on patients' fibroblasts demonstrated that the corresponding alleles carry null mutations of the DCLRE1C gene. Hum Mutat 30:1–11, 2010. © 2010 Wiley‐Liss, Inc.     

Sequence variation at the human ABO locus

The ABO blood group is the most important blood group system in transfusion medicine. Since the ABO gene was cloned and the molecular basis of the three major alleles delineated about 10 years ago, the gene has increasingly been examined by a variety of DNA‐based genotyping methods and analysed in detail by DNA sequencing. A few coherent observations emerge from these studies. First, there is extensive sequence heterogeneity underlying the major ABO alleles that produce normal blood groups A, B, AB and O when in correct combination with other alleles. Second, there is also extensive heterogeneity underlying the molecular basis of various alleles producing ABO subgroups such as A₂, A_x and B₃. There are over 70 ABO alleles reported to date and these alleles highlight the extensive sequence variation in the coding region of the gene. A unifying system of nomenclature is proposed to name these alleles. Third, extensive sequence variation is also found in the non‐coding region of the gene, including variation in minisatellite repeats in the 5′ untranslated region (UTR), 21 single nucleotide polymorphisms (SNPs) in intron 6 and one SNP in the 3′ UTR. The haplotypes of these variations reveal a specific relationship with the major ABO alleles. Fourth, excluding the common alleles, about half of the remaining alleles are due to new mutations and the other half can better be explained by intragenic recombination (both crossover and gene conversion) between common alleles. In particular, the recombination sites in hybrid alleles can be quite precisely defined through haplotype analysis of the SNPs in intron 6. This indicates that recombination is equally as important as point mutations in generating the genetic diversity of the ABO locus. Finally, a large number of ABO genotyping methods are available and are based on restriction analysis, allele specific amplification, mutation screening techniques or their combinations.     

Molecular characterization of banana virus X (BVX), a novel member of the <Emphasis Type="Italic">Flexiviridae</Emphasis> family

Summary. A novel virus was identified in banana (Musa spp). Analysis of the last 2917 nucleotides of its positive strand genomic RNA showed five open reading frames corresponding, from 5′ to 3′, to a truncated ORF coding for a replication-associated protein, three ORFs coding for a movement-associated triple gene block (TGB) and a capsid protein (CP) gene. This genome organization is similar to that of some members of the Flexiviridae family such as potexviruses and foveaviruses. This virus was named Banana virus X (BVX). Comparative sequence analysis showed that BVX is only distantly related to other members of the Flexiviridae family, in which it appears to define a new genus. BVX produces defective RNAs derived from its genomic RNA by non-homologous recombination. Three distinct pairs of donor/acceptor recombination sites involving short direct nucleotide repeats were characterized, accounting for deletions of 1268, 1358 and 1503 nucleotides. Contrary to the situation encountered for Potexviruses, these recombination sites are located within the TGB1 and CP genes and result in a truncated TGB1 protein.     

Novel marker for recombination in the 3′-untranslated region of members of the species Human enterovirus A

Human enterovirus A (HEV-A) is a species in the genus Enterovirus. Viruses belonging to this species are often responsible for hand, foot and mouth disease and associated acute neurological disease. Studies of the 3′ untranslated region (UTR) of human enterovirus 71 (HEV71) revealed a possible role in virus replication. We compared the 3′-UTRs of all members of HEV-A and confirmed the presence of a secondary structure comprising three stem-loop domains (SLDs). SLD-Z is situated closest to the stop codon and has been shown previously to affect plaque morphology. The prototype strains of coxsackieviruses A4 (CVA4), CVA14, and CVA16 carried the longer group I SLD-Z, whilst other CVAs and HEV71 carried the shorter group II SLD-Z. We demonstrate the importance of SLD-Z as a marker for the emergence of newer strains of HEV71 and CVA16 through inter-typic recombination and propose that SLD-Z is a novel evolutionary marker for recombination in HEV-A.     

Agrobacterium T-DNA-mediated integration and gene replacement in the brown rot pathogen Monilinia fructicola

A transformation system utilizing Agrobacterium tumefaciens was developed for targeted gene disruption in Monilinia fructicola, a fungal pathogen that causes brown rot disease of stone fruits. Transformation with a vector containing the neomycin phosphotransferase II (nptII) cassette flanked with 4 kb cutinase gene (Mfcut1) flanking sequences resulted in an average of 13 transformants per 10⁵ spores. When assayed by PCR and DNA blot analyses, more than 50% of the transformants recovered had integrated in the targeted Mfcut1 locus. Both target-gene-specific and non-specific integrations carried direct (head-to-tail) repeat T-DNA integrations. Sequence analysis of these T-DNA integrations revealed that 26 bp of the T-DNA right border were missing at the junctions between direct repeats in all cases. The recombination event during non-specific T-DNA integration in this fungus was unlike that reported in Agrobacterium-mediated transformation in plants.Note: Nucleotide sequence data reported are available in GenBank under the accession number–DQ173196.     

Molecular characterization of <Emphasis Type="Italic">Plum pox virus</Emphasis> Rec isolates from Russia suggests a new insight into evolution of the strain

Field isolates of Plum pox virus (PPV), belonging to the strain Rec, have been found for the first time in Russia. Full-size genomes of the isolates K28 and Kisl-1pl from myrobalan and plum, respectively, were sequenced on the 454 platform. Analysis of all known PPV-Rec complete genomes using the Recombination Detection Program (RDP4) revealed yet another recombination event in the 5′-terminal region. This event was detected by seven algorithms, implemented in the RDP4, with statistically significant P values and supported by a phylogenetic analysis with the bootstrap value of 87%. A putative PPV-M-derived segment, encompassing the C-terminus of the P1 gene and approximately two-thirds of the HcPro gene, is bordered by breakpoints at positions 760–940 and 1838–1964, depending on the recombinant isolate. The predicted 5′-distal breakpoint for the isolate Valjevka is located at position 2804. The Dideron (strain D) and SK68 (strain M) isolates were inferred as major and minor parents, respectively. Finding of another recombination event suggests more complex evolutionary history of PPV-Rec than previously assumed. Perhaps the first recombination event led to the formation of a PPV-D variant harboring the PPV-M-derived fragment within the 5′-proximal part of the genome. Subsequent recombination of its descendant with PPV-M in the 3′-proximal genomic region resulted in the emergence of the evolutionary successful strain Rec.     

A novel heterozygous mutation in the Indian hedgehog gene (IHH) is associated with brachydactyly type A1 in a Chinese family

Brachydactyly type A1 (BDA1) is caused by mutations in the Indian hedgehog gene, IHH, on chromosome 2q35-36. In this study, a large five-generation Chinese family with BDA1 was identified and characterized. All affected family members demonstrated significant homogeneous phenotype and some unique clinical features different from those associated with the reported BDA1 mutations in IHH. Linkage analysis showed that the BDA1 gene in the family was linked to marker D2S126 close to IHH with a LOD score of 4.74 at a recombination fraction of 0. DNA sequence analysis revealed a heterozygous C to T transition at nucleotide 461 of IHH, resulting in a novel T154I substitution. The T154I mutation co-segregated with all affected individuals in the family, and was not present in normal family members or 200 normal controls. These results expand the spectrum of clinical phenotype associated with IHH mutations.M. Liu and X. Wang contribute equally to this work     

Breakpoint clusters of the PML gene in acute promyelocytic leukemia: Primary structure of the reciprocal products of the PML-RARA gene in a patient with t(15;17)

DNA studies of the translocation t(15;17) in acute promyelocytic leukemia (APL) have shown that the retinoic acid receptor alpha (RARA) gene on chromosome 17 is juxtaposed to the promyelocytic leukemia (PML) gene on chromosome 15. The PML breakpoints have been mapped to 3 clusters: bcr1, bcr2, and bcr3. We have examined the PML breakpoint distribution in a series of 33 Chinese patients with APL Twenty-two patients fell within bcr1, 2 within bcr2, and 9 within bcr3. The primary structure of the reciprocal chromosome translocation joints of one patient and that of their normal counterparts have been determined and compared to those of 2 previously reported cases. These studies revealed possible topoisomerase II cleavage sites close to the breakpoints and suggested implications of DNA attachment sites to nuclear matrix. We propose that these features are relevant to the process of illegitimate recombination generating the translocation. © 1993 Wiley-Liss, Inc.     

Woodchuck hepatitis virus DNA integration in a common chromosomal region of the woodchuck genome in two independent hepatocellular carcinomas

Summary.  In woodchuck hepatocellular carcinoma (HCC) the myc-oncogene family (particularly N-myc2) and the win locus of cellular genome have been reported as frequent targets for integration of woodchuck hepatitis virus (WHV) DNA. In this paper a further cellular locus, b3n, is reported as recurrent target for WHV integration in woodchuck HCC. Cloning and sequencing of a WHV-DNA integration and its cellular flanking regions showed that viral DNA was inserted in a chromosomal region already described for WHV integration in another single HCC. The two integration sites are only 0.5 kb apart. A link between WHV integration in b3n and HCC development may be postulated. Careful analysis of the sequence of the unoccupied locus revealed that, in addition to Alu-like repeats and a gag-like coding region, already described, several features of Matrix Attachment Region (MAR) sequences are present. Thus (part of) b3n might be a previously unrecognized MAR. Organization of the chromatin in functional domains and regulation of gene expression are some functions attributed to MAR sequences. The occurrence of WHV-DNA integration close to the same putative MAR in two different HCCs suggests that a mechanism of deregulation of MAR functions by WHV insertion might act in some liver tumors.     

Identification and characterization of a new conjugation/type IVA secretion system (trb/tra) of Legionella pneumophila Corby localized on two mobile genomic islands

Horizontal gene transfer probably contributes to evolution of Legionella pneumophila and its adaptation to different environments. Although horizontal gene transfer was observed in Legionella, the mechanism is still not specified. In this study we identified and analysed a new type of conjugation/type IVA secretion system (trb/tra) of L. pneumophila Corby, a virulent human isolate. Two similar versions of this conjugation system were identified, localized on two different genomic islands (Trb-1, 42,710 bp and Trb-2, 34,434 bp). Trb-1 and Trb-2 are integrated within the tRNA^Pro gene (lpc2778) and the tmRNA gene (lpc0164), respectively. Both islands exhibit an oriT region and both can be excised from the chromosome forming episomal circles. Trb-1 was analysed in more detail. It is active and can be horizontally transferred to other Legionella strains by conjugation and then integrated into the genome in a site-specific manner within the tRNA^Pro gene. We characterized the sequence of the excision and integration sites of Trb-1 in three different L. pneumophila strains. Here we demonstrate that L. pneumophila exhibits a functional oriT region and that genomic islands in Legionella can be mobilized and conjugated to other species of Legionella. Thus, we describe for the first time a mechanism that may explain the observed horizontal transfer of chromosomal DNA in Legionella.     

Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′′-monophosphate decarboxylase

A homologous transformation system for the opportunistic fungal pathogen Aspergillus fumigatus was developed. It is based on the A. fumigatus pyrG gene, encoding orotidine 5′-monophosphate decarboxylase, which was cloned and sequenced. Transformation of both Aspergillus (Emericella) nidulans and A. fumigatus pyrG mutant strains by the use of protoplasts or electroporation established the functionality of the cloned gene. DNA sequencing of the A. fumigatus pyrG1 mutant allele revealed that it encodes a truncated, non-functional, PyrG protein. Transformation of an A. fumigatus pyrG1 mutant with a plasmid carrying the novel pyrG2 allele constructed by in vitro mutagenesis yielded prototrophic transformants following recombination between both mutation sites. Analysis of transformants carrying the entire plasmid showed that up to 45% of integration had occurred at the pyrG locus. This provides a tool to target defined genetic constructs at a specific locus in the A. fumigatus genome in order to study gene regulation and function. Received: 12 December 1997 / 11 March 1998     

Genomic sequence analysis of virulent Cronobacter sakazakii bacteriophage ES2

Virulent Cronobacter sakazakii bacteriophage ES2 was isolated from swine fecal samples, and the genome sequence by was determined GS-Flx. Bacteriophage ES2 had a double-stranded DNA genome with a length of 22,162 bp and a G+C content of 50.08%. The morphological characteristics under a transmission electron microscope indicated that bacteriophage ES2 belongs to the family Myoviridae. The structural proteins, including the phage coat protein, were separated by SDS-PAGE and identified by Q-TOF. Bioinformatics analysis of the bacteriophage genome revealed 30 putative open reading frames (ORFs). The predicted protein products of the ORFs were determined and described. To our knowledge, the genome of the newly isolated bacteriophage ES2 was not significantly similar to that of any previously reported bacteriophages of members of the family Enterobacteriaceae.     

High frequency recombination betweenloxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase

An adenovirus vector (AdCre1) expressing Cre recombinase has been used to induce recombination betweenloxP sites in human chromosomes. G418 resistant cells with oneloxP site, generated by transfection with a plasmid containingloxP between the SV40 promoter and the G418 resistance (neo) gene, were infected with AdCre1 and transfected with a plasmid containingloxP adjacent to a promoterless hisD gene. This resulted in integration of hisD downstream of the SV40 promoter with gain of histidinol and loss of G418 resistance. Since AdCre1 is non-replicating and Cre expression transient, histidinol resistant cells containing the hisD gene flanked byloxP sites were stable. Reinfection of these cells with AdCre1 induced excision of hisD in over 90% of infected cells. This high efficiency of site-specific recombination suggests that AdCre1 may be exploited for temporal and tissue-specific regulation of gene expression and for chromosome engineering in vitro and in animals.     

3′ End structure and rearrangements of EGFR in glioblastomas

Rearrangements of EGFR are known to occur in a significant fraction of glioblastomas, the most common and malignant form of central nervous system tumor. Although the consequences of these alterations have been described at the mRNA and protein level, little is known about human EGFR genomic sequence or organization at the rearrangement sites. To investigate one group of alterations in glioblastoma, we used long-range PCR to synthesize a segment of the gene near its 3′ end, which is frequently rearranged in tumors with EGFR amplification. The sequence of this PCR product provided a precise map for the five 3′-terminal exons of EGFR, designated as exons 22–26. Ten tumors were identified with rearrangements in this part of the gene, most of which resulted in the loss of 325 coding bases that constitute exons 23–25. No two tumors shared identical donor or acceptor rearrangement sites, and the examination of sequences at these sites failed to support homologous recombination as a mechanism responsible for any of the rearrangements. However, examination of the entire exon 22–26 region for sequence motifs associated with genomic instability identified two large, CA-rich tracts in intron 25. Genes Chromosomes Cancer 23:248–254, 1998. © 1998 Wiley-Liss, Inc.     

Genetic recombination in human melanoma and astrocytoma cell lines involves oncogenes and growth factor genes

We describe here the chromosomal distribution of sister chromatid exchanges (SCEs) in four human tumor cell lines (two melanomas and two astrocytomas), and have mapped the sister chromatid recombination (SCR) sites. A higher incidence of SCR sites than expected on the basis of chromosome length occurred in chromosomes 2, 4, 5 and 15 in both the RPMI 5966 and MEL57 melanoma cell lines, and in chromosomes 1, 5, 13 and 15 of the IJKt and GUVW astrocytoma cell lines. A majority of the recombination sites occurred close to chromosomal fragile sites. A third of these occurred at the same bands as fragile sites. The recombination sites involved the N-ras and the epidermal growth factor gene in the melanomas. In the astrocytomas, the N-ras, Rb and c-mos genes appeared to be involved in the recombination events. The β2-microglobulin gene was involved in both astrocytomas and one melanoma. The erb132 was involved in SCR only in the RPMI melanoma.