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PubMed1999

[Genome analysis with restriction endonucleases recognizing 4- or 5-base pair sequences of adenovirus type 4].

PURPOSE: To ascertain the chronological change of subgenome types of adenovirus type 4 (Ad 4) through DNA analysis. MATERIALS AND METHODS: We evaluated sixteen Ad 4 strains from patients with acute viral conjunctivitis at four eye clinics in Sapporo, northern Japan. Seven strains were obtained from December 1993 through March 1994 (first period). Nine strains were obtained from March through May in 1995 (second period). These strains were analysed using DNA restriction endonucleases recognizing 4- or 5-base-pair sequences, Taq I and Hin f I. RESULTS: The genome type of sixteen strains was Ad 4 a. Seven strains in the first period showed the same subgenome type. Nine strains in the second period showed two subgenome types: five strains showing the same subgenome type as in the first period, and four showing a different subgenome type. The two subgenome types were similar in DNA pattern, suggesting that both were derivatives of a common subgenome type. All the sixteen strains were a new subgenome type which was different from those previously isolated during 1985-1989. CONCLUSION: The subgenome types of Ad 4 in 1993-1995 have changed from those in 1985-1989.

Adenoviruses, Human↗
PubMed2000

A homokaryotic derivative of a Phanerochaete chrysosporium strain and its use in genomic analysis of repetitive elements.

Analysis of complex gene families in the lignin-degrading basidiomycete Phanerochaete chrysosporium has been hampered by the dikaryotic nuclear condition. To facilitate genetic investigations in P. chrysosporium strain BKM-F-1767, we isolated a homokaryon from regenerated protoplasts. The nuclear condition was established by PCR amplification of five unlinked genes followed by probing with allele-specific oligonucleotides. Under standard nitrogen-limited culture conditions, lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities of the homokaryon were equivalent to those of the parental dikaryon. We used the homokaryon to determine the genomic organization and to assess transcriptional effects of a family of repetitive elements. Previous studies had identified an insertional mutation, Pce1, within lignin peroxidase allele lipI2. The element resembled nonautonomous class II transposons and was present in multiple copies in strain BKM-F-1767. In the present study, three additional copies of the Pce1-like element were cloned and sequenced. The distribution of elements was nonrandom; all localized to the same 3.7-Mb chromosome, as assessed by segregation analysis and Southern blot analysis of the homokaryon. Reverse transcription-PCR (RT-PCR) showed that Pce1 was not spliced from the lipI2 transcript in either the homokaryon or the parental dikaryon. However, both strains had equivalent lignin peroxidase activity, suggesting that some lip genes may be redundant.

Chromosome Mapping↗
PubMed2006

Genomic analysis of the rhg1 locus: candidate genes that underlie soybean resistance to the cyst nematode.

The rhg1 gene or genes lie at a recessive or co-dominant locus, necessary for resistance to all Hg types of the soybean (Glycine max (L.) Merr.) cyst nematode (Heterodera glycines I.). The aim here was to identify nucleotide changes within a candidate gene found at the rhg1 locus that were capable of altering resistance to Hg types 0 (race 3). A 1.5 +/- 0.25 cM region of chromosome 18 (linkage group G) was shown to encompass rhg1 using recombination events from four near isogenic line populations and nine DNA markers. The DNA markers anchored two bacterial artificial chromosome (BAC) clones 21d9 and 73p6. A single receptor like kinase (RLK; leucine rich repeat-transmembrane-protein kinase) candidate resistance gene was amplified from both BACs using redundant primers. The DNA sequence showed nine alleles of the RLK at Rhg1 in the soybean germplasm. Markers designed to detect alleles showed perfect association between allele 1 and resistance to soybean cyst nematode Hg types 0 in three segregating populations, fifteen additional selected recombination events and twenty-two Plant Introductions. A quantitative trait nucleotide (QTN) [corrected] in the RLK at rhg1 was inferred that alters A87 to V87 in the context of H274 rather than N274. [corrected] Contiguous DNA sequence of 315 kbp of chromosome 18 (about 2 cM) contained additional gene candidates that may modulate resistance to other Hg-types including a variant laccase, a hydrogen-sodium ion antiport and two proteins of unknown function. A molecular basis for recessive and co-dominant resistance that involves interactions among paralagous disease-resistance genes was inferred that would improve methods for developing new nematode-resistant soybean cultivars.

Animals↗
PubMed2003

Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes.

We analyzed the breadth of the unfolded protein response (UPR) in Arabidopsis using gene expression analysis with Affymetrix GeneChips. With tunicamycin and DTT as endoplasmic reticulum (ER) stress-inducing agents, we identified sets of UPR genes that were induced or repressed by both stresses. The proteins encoded by most of the upregulated genes function as part of the secretory system and comprise chaperones, vesicle transport proteins, and ER-associated degradation proteins. Most of the downregulated genes encode extracellular proteins. Therefore, the UPR may constitute a triple effort by the cell: to improve protein folding and transport, to degrade unwanted proteins, and to allow fewer secretory proteins to enter the ER. No single consensus response element was found in the promoters of the 53 UPR upregulated genes, but half of the genes contained response elements also found in mammalian UPR regulated genes. These elements are enriched from 4.5- to 15-fold in this upregulated gene set.

Arabidopsis↗
PubMed2004

Genomic analysis of essentiality within protein networks.

In this article, we introduce the notion of 'marginal essentiality' through combining quantitatively the results from large-scale phenotypic experiments (e.g. growth rate inhibition from knockouts). We find that this quantity relates to many of the topological characteristics of protein-protein interaction networks. In particular, proteins with a greater degree of marginal essentiality tend to be network hubs (i.e. with many interactions) and tend to have a shorter characteristic path length to their neighbors. We extend our network analysis to encompass transcriptional regulatory networks. Although transcription factors with many targets tend to be essential, surprisingly, we find that genes that are regulated by many transcription factors are usually not essential.

Animals↗
PubMed2004

Functional genomic analysis of the ADP-ribosylation factor family of GTPases: phylogeny among diverse eukaryotes and function in C. elegans.

ADP-ribosylation factor (Arf) and Arf-like (Arl) proteins are a family of highly conserved 21 kDa GTPases that emerged early in the evolution of eukaryotes. These proteins serve regulatory roles in vesicular traffic, lipid metabolism, microtubule dynamics, development, and likely other cellular processes. We found evidence for the presence of 6 Arf family members in the protist Giardia lamblia and 22 members in mammals. A phylogenetic analysis was performed to delineate the evolutionary relationships among Arf family members and to attempt to organize them by both their evolutionary origins and functions in cells and/or organisms. The approximately 100 protein sequences analyzed from animals, fungi, plants, and protists clustered into 11 groups, including Arfs, nine Arls, and Sar proteins. To begin functional analyses of the family in a metazoan model organism, we examined roles for all three C. elegans Arfs (Arf-1, Arf-3, and Arf-6) and three Arls (Arl-1, Arl-2, and Arl-3) by use of RNA-mediated interference (RNAi). Injection of double-stranded RNA (dsRNA) encoding Arf-1 or Arf-3 into N2 hermaphrodites produced embryonic lethality in their offspring and, later, sterility in the injected animals themselves. Injection of Arl-2 dsRNA resulted in a disorganized germline and sterility in early offspring, with later offspring exhibiting an early embryonic arrest. Thus, of the six Arf family members examined in C. elegans, at least three are required for embryogenesis. These data represent the first analysis of the role(s) of multiple members of this family in the development of a multicellular organism.

ADP-Ribosylation Factors↗
PubMed2003

Utility of comparative anchor-tagged sequences as physical anchors for comparative genome analysis among the Culicidae.

The development of comparative genetic maps in multiple species of mosquitoes could prove extremely useful in the search for those genes that contribute to mosquito vector competence or genes associated with other phenotypes of interest. To effectively compare these gene maps, markers must be developed that are based on chromosomal regions conserved throughout the Culicidae. We designed 35 polymerase chain reaction (PCR) primer pairs based upon orthlogous exons in Aedes aegypti and Drosophila melanogaster or Anopheles gambiae. Twenty-three of the primers yielded a single PCR product in at least one dipteran, in addition to Ae. aegypti, when screened with genomic DNA from seven dipterans, including five mosquito species. Eight of the primers amplified a single PCR product in only Ae. aegypti, while four primer pairs gave no PCR product in any species. The 23 successful comparative anchor-tagged sequence primer pairs give broad genome coverage in Ae. aegypti, and more importantly demonstrate an efficient strategy for developing comparative anchor marker loci for any species of Culicidae.

Amino Acid Sequence↗
PubMed2001

Genome analysis of Agrobacterium tumefaciens: construction of physical maps for linear and circular chromosomal DNAs, determination of copy number ratio and mapping of chromosomal virulence genes.

The phytopathogenic bacterium Agrobacterium tumefaciens is unique in that it possesses both linear and circular DNA chromosomes in addition to a plant-tumor-inducing (Ti) plasmid. We analyzed the two chromosomal DNA molecules in strain MAFF301001, whose Ti plasmid has already been sequenced completely. Physical maps of the chromosomal DNAs were constructed by Southern hybridization experiments using Pme I and Swa I fragments and short fragments bridging the Swa I fragments with special care to avoid any missing fragment. Hybridization with 16S rDNA probe showed one rDNA locus on the linear chromosome and two loci on the circular chromosome. For this bacterium to be pathogenic, not only Ti plasmid but also chromosomal genes are required. The chromosomal virulence (chv) genes (chvA, chvB, chvD, chvE, chvG, chvH, and chvI) and the chromosomal genes affecting the virulence [acvB, pgm(exoC), glgP, miaA, and ros] were successfully mapped onto 5 different regions in the chromosomal physical maps. These chv genes and the chromosomal genes affecting the virulence other than pgm and glgP were found on the circular chromosome, whereas the pgm and glgP genes were located on the linear chromosome. In contrast to the large terminal inverted repeats of Streptomyces linear chromosomal DNA, no hybridization signal was detected between left and right terminal fragments of the linear A. tumefaciens chromosome. Quantitative analysis of DNA fragments indicated that the copy numbers of the two chromosomal DNAs and the Ti plasmid are identical.

Agrobacterium tumefaciens↗
PubMed2004

Characterization and comparative genomic analysis of intronless Adams with testicular gene expression.

ADAM (a disintegrin and metalloprotease) family members with testis-specific or -predominant gene expression are divided phylogenically into two groups: ADAMs 2, 3, 5, 27, and 32 (the first group) and ADAMs 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the second group). We cloned and sequenced cDNAs for previously unidentified mouse Adams that belong to the second group. We found that all the Adam genes in the second phylogenic group are transcribed by both somatic and germ cells in mouse testis, representing a unique expression pattern different from that of the first-group Adams. Genomic analyses revealed that all the second-group Adam genes lack introns interrupting protein-coding sequences and many of them are present as multicopy genes, resulting in total of 14 functional mouse genes in this phylogenic group. Comparing the mouse and human ADAM genes, we found that a number of these mouse Adam genes do not have human orthologues and, even if they exist, some orthologues are pseudogenes in human. These results suggest the differential expansion of the second-group Adam genes in the mouse genome during evolution and a relationship between these Adams and male reproduction unique to mouse.

Amino Acid Sequence↗
PubMed2002

Global genomic analysis of AlgU (sigma(E))-dependent promoters (sigmulon) in Pseudomonas aeruginosa and implications for inflammatory processes in cystic fibrosis.

The conversion of Pseudomonas aeruginosa to the mucoid phenotype coincides with the establishment of chronic respiratory infections in cystic fibrosis (CF). A major pathway of conversion to mucoidy in clinical strains of P. aeruginosa is dependent upon activation of the alternative sigma factor AlgU (P. aeruginosa sigma(E)). Here we initiated studies of AlgU-dependent global expression patterns in P. aeruginosa in order to assess whether additional genes, other than those involved in the production of the mucoid exopolysaccharide alginate, are turned on during conversion to mucoidy. Using genomic information and the consensus AlgU promoter sequence, we identified 35 potential AlgU (sigma(E)) promoter sites on the P. aeruginosa chromosome. Each candidate promoter was individually tested by reverse transcription and mRNA 5'-end mapping using RNA isolated from algU(+) and algU::Tc(r) mutant cells. A total of 10 new AlgU-dependent promoters were identified, and the corresponding mRNA start sites were mapped. Two of the 10 newly identified AlgU promoters were upstream of predicted lipoprotein genes. Since bacterial lipoproteins have been implicated as inducers of inflammatory pathways, we tested whether lipopeptides corresponding to the products of the newly identified AlgU-dependent lipoprotein genes, lptA and lptB, had proinflammatory activity. In human peripheral blood monocyte-derived macrophages the peptides caused production of interleukin-8, a proinflammatory chemokine typically present at excessively high levels in the CF lung. Our studies show how genomic information can be used to uncover on a global scale the genes controlled by a given sigma factor (collectively termed here sigmulon) using conventional molecular tools. In addition, our data suggest the existence of a previously unknown connection between conversion to mucoidy and expression of lipoproteins with potential proinflammatory activity. This link may be of significance for infections and inflammatory processes in CF.

Amino Acid Sequence↗
PubMed2005

Genome analysis and gene expression profiling of neuroblastoma and ganglioneuroblastoma reveal differences between neuroblastic and Schwannian stromal cells.

Neuroblastic tumours are a group of paediatric cancers with marked morphological heterogeneity. Neuroblastoma (Schwannian stroma-poor) (NB-SP) is composed of undifferentiated neuroblasts. Ganglioneuroblastoma intermixed (Schwannian stroma-rich) (GNBi-SR) is predominantly composed of Schwannian stromal (SS) and neuroblastic (Nb) cells. There are contrasting reports suggesting that SS cells are non-neoplastic. In the present study, laser capture microdissection (LCM) was employed to isolate SS and Nb cells. Chromosome 1p36 deletion and MYCN gene amplification were found to be associated in two out of seven NB-SPs, whereas no abnormalities were observed in five GNBi-SRs. In some cases, loss of heterozygosity (LOH) at 1p36 loci was detected in Nb cells but not in the bulk tumour by LCM; furthermore, LOH was also identified in both SS and tumour tissue of a GNBi-SR. DNA gain and loss studied by comparative genomic hybridization were observed at several chromosome regions in NB-SP but in few regions of GNBi-SR. Finally, gene expression profiles studied using an oligo-microarray technique displayed two distinct signatures: in the first, 32 genes were expressed in NB-SP and in the second, 14 genes were expressed in GNBi-SR. The results show that NB-SP is composed of different morphologically indistinguishable malignant cell clones harbouring cryptic mutations that are detectable only after LCM. The degree of DNA imbalance is higher in NB-SP than in GNBi-SR. However, when the analysis of chromosome 1p36 is performed at the level of microdissection, LOH is also observed in SS cells. These data provide supportive evidence that SS cells have a less aggressive phenotype and play a role in tumour maturation.

Chromosomes, Human, Pair 1↗
PubMedDate not supplied

Coincident sequence cloning: a new approach to genome analysis.

Many analytical molecular genetic techniques developed over the past decade have evolved primarily to tackle the problem of targeting or isolating sequences of interest in complex mixtures. A new approach to this problem, Coincident Sequence Cloning (CSC), involves integrating a pair of DNA mixtures in such a way as to isolate any shared sequence components.

Cloning, Molecular↗
PubMed2001

Genomic analysis of MIC genes in rhesus macaques.

MIC genes map to the major histocompatibility complex (MHC) and are distantly related to MHC class I genes. Recently, MICA/MICB-like genes have been described in nonhuman primates. In Macaca mulatta, three MICA/B-like genes could be identified: Mamu-MIC1, Mamu-MIC2, and Mamu-MIC3. We show here the isolation and characterization of rhesus macaque cosmid clones which carry the Mamu-MIC2 and Mamu-MIC3 genes. Neither the MIC2- and MIC3-coding sequences nor respective flanking sequences can be aligned unambiguously to either the human HLA-MICA or -MICB subregions, although MIC2 was found at a similar distance to the BAT1 gene as known for MICB in human. Thus, the characteristics allowing for a classification of primate MIC genes as being of the MICA or MICB types appear to have evolved after the separation of humans and rhesus monkeys from a common ancestor. Furthermore, also Mamu-MICD-containing cosmids could be isolated. In contrast to Mamu-MIC2 and Mamu-MIC3, the Mamu-MICD gene and its flanking sequences are highly conserved and orthologous to the human MICD subregion.

Animals↗
PubMed2007

Genomic analysis of the 8p11-12 amplicon in familial breast cancer.

Amplification of 8p11-12 has been recurrently reported in sporadic breast cancer. These studies define a complex molecular structure with a set of minimal amplified regions, and different putative oncogenes that show a strong correlation between amplification and over-expression such as ZNF703/FLJ14299, SPFH2/C8orf2, BRF2 and RAB11FIP. However, none of these studies were carried out on familial breast malignancies. We have studied the incidence, molecular features and clinical value of this amplification in familial breast tumors associated with BRCA1, BRCA2 and non-BRCA1/2 gene mutations. We detected 9 out of 80 familial tumors with this amplicon by chromosomal comparative genomic hybridization. Next, we used a high-resolution comparative genomic hybridization array covering the 8p11-12 region to characterize this chromosomal region. This approach allowed us to define 2 cores of common amplification that largely overlap with those reported in sporadic tumors. Our findings confirm the molecular complexity of this chromosomal region and indicate that this genomic event is a common alteration in breast cancer, present not only in sporadic but also in familial tumors. Finally, we found correlation between the 8p11-12 amplification and proliferation (Ki-67) and cyclin E expression, which further proves in familial tumors the poor prognosis association previously reported in sporadic breast cancer.

Adult↗
PubMed2001

Genomic analysis and localization of murine Deltex, a modulator of notch activity, to mouse chromosome 5 and its human homolog to chromosome 12.

Deltex is a component of the Notch signaling network, which mediates cellular differentiation, proliferation, and apoptosis during development. Murine Deltex was initially isolated as a cDNA transcript that displayed increased expression in T-cell tumors induced by gamma irradiation. The in vivo function of Deltex is unknown; however, the emerging role of Notch signaling in T-cell development and lymphomagenesis indirectly supports a role for Deltex in these processes. To investigate the regulation of Deltex expression in both normal and transformed tissue, we have begun analyzing the Deltex genomic locus. Here, we report the exon-intron organization of Deltex and map the locus to the middistal region of mouse chromosome 5, tightly linked to the Adam1a, Lnk, Tbx5, and Nos1 loci. The human homolog of Deltex has been localized to chromosome 12.

3' Untranslated Regions↗
PubMed2006

Full genomic analysis of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission.

The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.

Amino Acid Sequence↗
PubMed2019

Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains.

How pathogens evolve their virulence to humans in nature is a scientific issue of great medical and biological importance. Shiga toxin (Stx)-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are the major foodborne pathogens that can cause hemolytic uremic syndrome and infantile diarrhea, respectively. The locus of enterocyte effacement (LEE)-encoded type 3 secretion system (T3SS) is the major virulence determinant of EPEC and is also possessed by major STEC lineages. Cattle are thought to be the primary reservoir of STEC and EPEC. However, genome sequences of bovine commensal E. coli are limited, and the emerging process of STEC and EPEC is largely unknown. Here, we performed a large-scale genomic comparison of bovine commensal E. coli with human commensal and clinical strains, including EPEC and STEC, at a global level. The analyses identified two distinct lineages, in which bovine and human commensal strains are enriched, respectively, and revealed that STEC and EPEC strains have emerged in multiple sublineages of the bovine-associated lineage. In addition to the bovine-associated lineage-specific genes, including fimbriae, capsule, and nutrition utilization genes, specific virulence gene communities have been accumulated in stx- and LEE-positive strains, respectively, with notable overlaps of community members. Functional associations of these genes probably confer benefits to these E. coli strains in inhabiting and/or adapting to the bovine intestinal environment and drive their evolution to highly virulent human pathogens under the bovine-adapted genetic background. Our data highlight the importance of large-scale genome sequencing of animal strains in the studies of zoonotic pathogens.

Animals↗
PubMed2003

Genomic analysis of a recently identified virus (SEN virus) and genotypes D and H by polymerase chain reaction.

BACKGROUND/AIMS: SEN virus (SENV) was discovered in 1999 as a DNA virus with hepatotropic properties. Nine genotypes (A-I) have been identified with genotypes D and H being more prevalent in cases of chronic hepatitis. Attempts to determine whether SENV causes liver disease have been hampered by limited diagnostic testing. METHODS: In the present study, we developed two PCR based assays; a general SENV screening and genotype-specific assay. RESULTS: By screening PCR, the specificity for all SENV genotypes and SENV-related sequences was 20/20 (100%) with confirmation of the results being provided by genomic sequencing. With the genotype-specific PCR, specificities for SENV-D and SENV-H were 7/7 (100%) and 7/11 (64%), respectively. All screening PCR products were cloned and sequenced. The results of sequencing showed high genetic diversity in representative SENV genotypes. Five of twenty patients (25%) had mixed infections with several SENV genotypes. CONCLUSIONS: The screening PCR was useful for identifying cases of SENV infection. However, because of high genetic divergence and mixed co-infection, it was difficult to establish a specific method for genotype distinction. Hence, sequencing is still required for further investigations of SENV as a potential cause of liver disease.

Amino Acid Sequence↗