Biomedical subjects

Genomics

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PubMed2001

Comparative analysis of the genomic organization of Pax9 and its conserved physical association with Nkx2-9 in the human, mouse, and pufferfish genomes.

As a first step towards the identification of cis-regulatory elements of Pax9 by means of comparative genomics, we have analyzed genome regions encompassing the Pax9 gene in three vertebrate species, humans, mice (Mus musculus), and the Japanese pufferfish (Fugu rubripes). We show the genomic organization of Pax9 and its physical association with Nkx2-9 conserved in the three species. We discuss about possible implications of the conserved synteny between Pax9 and Nkx2-9 in a context of vertebrate evolution. This report also includes the first description of the primary structures of Fugu Pax9 and Nkx2-9. Furthermore, we report the identification of a novel upstream exon and putative transcription start sites in mouse Pax9. Our results suggest that transcription of Pax9 may be initiated at two alternative start sites and driven by TATA-less promoters.

Amino Acid Sequence↗

PubMed1996

Detection of amplified genomic sequences in human small-cell lung carcinoma cells by arbitrarily primed-PCR genomic fingerprinting.

The arbitrarily primed-PCR (AP-PCR) genomic fingerprinting method was applied to evaluate its effectiveness in detecting and characterizing amplified DNA fragments in two small-cell lung carcinoma (SCLC) cell lines, NCI-H69 and NCI-H82. Of the 2428 DNA fragments detected by AP-PCR using 62 arbitrary primers, 2 (0.08%) DNA fragments were amplified in NCI-H69 and 6 (0.25%) DNA fragments were amplified in NCI-H82. Based on these results, we estimate the total size of the amplified genomic regions in these cell lines to be 3000 megabase pairs (Mb) x 0.0008 = 2.4 Mb in NCI-H69 and 3000 Mb x 0.0025 = 7.5 Mb in NCI-H82. The 2 amplified fragments in NCI-H69 were mapped to chromosome 2, and all 6 amplified fragments in NCI-H82 were mapped to chromosome 8. This strongly suggests that restricted chromosomal regions are specifically amplified in these SCLC cell lines. Since the N-myc gene at 2p24 is amplified in NCI-H69 and the c-myc gene at 8q24 is amplified in NCI-H82, it is possible that these DNA fragments are co-amplified with N-myc or c-myc in these cell lines. However, since the 2 amplified fragments in NCI-H69 were not amplified in 42 other human cancer cell lines including 11 cell lines carrying amplified N-myc genes, it is also possible that there are amplified regions on chromosome 2 other than the N-myc locus at 2p24 in NCI-H69. In contrast, all 6 amplified fragments in NCI-H82 were amplified in several other human cancer cell lines carrying amplified c-myc genes. This result further indicates that these fragments were derived from an amplification unit that includes the c-myc gene. Our results show the ability of the AP-PCR method to analyze the fraction of the genome with amplification in human cancer cells.

Base Sequence↗

PubMed1985

Nucleotide sequence and genetic organization of the genome of the N-specific filamentous bacteriophage IKe. Comparison with the genome of the F-specific filamentous phages M13, fd and f1.

The nucleotide sequence and genetic organization of the genome of the N-specific filamentous single-stranded DNA phage IKe has been established and compared with that of the F-specific filamentous phages M13, fd and f1 (Ff). The IKe DNA sequence comprises 6883 nucleotides, which is 476 (475) nucleotides more than the nucleotide sequence of the Ff genome. The data indicate that IKe and Ff have evolved from a common ancestor (overall homology approx. 55%) and that their genomes contain ten homologous genes, the order of which is identical. Similar to Ff, the major coat protein and the gene III-encoded pilot protein of IKe are synthesized via precursor molecules. The extent of homology between the genes of IKe and Ff differs significantly from one gene to another. Genes that code for viral capsid proteins are less homologous than genes whose products are involved in the processes of DNA replication and phage morphogenesis. During evolution, large nucleotide sequence rearrangements have occurred in the gene (gene III) whose product is needed for the attachment of the virion to the conjugative pili of the host cell, suggesting that these rearrangements have led to phages with different host specificities. Extensive nucleotide sequence homology was noted between the structural elements involved in DNA replication and phage morphogenesis, indicating that the mechanisms involved in DNA replication and morphogenesis are highly conserved.

Base Sequence↗

PubMed1987

Tumorigenic poxviruses: genomic organization and DNA sequence of the telomeric region of the Shope fibroma virus genome.

Shope fibroma virus (SFV), a tumorigenic poxvirus, has a 160-kb linear double-stranded DNA genome and possesses terminal inverted repeats (TIRs) of 12.4 kb. The DNA sequence of the terminal 5.5 kb of the viral genome is presented and together with previously published sequences completes the entire sequence of the SFV TIR. The terminal 400-bp region contains no major open reading frames (ORFs) but does possess five related imperfect palindromes. The remaining 5.1 kb of the sequence contains seven tightly clustered and tandemly oriented ORFs, four larger than 100 amino acids in length (T1, T2, T4, and T5) and three smaller ORFs (T3A, T3B, and T3C). All are transcribed toward the viral hairpin and almost all possess the consensus sequence TTTTTNT near their 3' ends which has been implicated for the transcription termination of vaccinia virus early genes. Searches of the published DNA database revealed no sequences with significant homology with this region of the SFV genome but when the protein database was searched with the translation products of ORFs T1-T5 it was found that the N-terminus of the putative T4 polypeptide is closely related to the signal sequence of the hemagglutinin precursor from influenza A virus, suggesting that the T4 polypeptide may be secreted from SFV-infected cells. Examination of other SFV ORFs shows that T1 and T2 also possess signal-like hydrophobic amino acid stretches close to their N-termini. The protein database search also revealed that the putative T2 protein has significant homology to the insulin family of polypeptides. In terms of sequence repetitions, seven tandemly repeated copies of the hexanucleotide ATTGTT and three flanking regions of dyad symmetry were detected, all in ORF T3C. A search for palindromic sequences also revealed two clusters, one in ORF T3A/B and a second in ORF T2. ORF T2 harbors five short sequence domains, each of which consists of a 6-bp short palindrome and a 10- to 18-bp larger palindrome. The significance of these palindromic domains in this ORF is unclear but the coincidence of the end of one larger palindrome with the end of the translated protein sequence that has homology with the B chain of insulin suggests that the palindromes may divide the T2 protein into several functional units. The salient organizational features of the complete SFV TIR are also discussed in light of what is known about other poxviral TIRs.

Amino Acid Sequence↗

PubMed1979

Genome analysis of Amphioxus and speculation as to the origin of contrasting vertebrate genome organization patterns.

1. The genome of Amphioxus was investigated by DNA reassociation techniques for the amount of repetitive and non-repetitive sequences and its pattern of organization. 2. A comparison of the amount of non-repetitive DNA between Amphioxus and the tunicate Ciona intestinalis does not support the hypothesis that the Cephalochordates have arisen from the Tunicates by polyploidy. 3. In the Amphioxus genome repetitive and non-repetitive elements are predominantly arranged in a short period interspersion pattern. Conclusions are presented as to the evolution of contrasting genome organization patterns among vertebrates.

Animals↗

PubMed1979

Amount of repeated and non-repeated DNA in the genomes of closely related fish species with varying genome sizes.

1. Within the teleostean family Cyprinidae, diploid species occur with wide variation in genome size. There also exist species which were anciently tetraploid. 2. The quantitative changes of DNA content in the diploids are primarily due to differences in the amount of intermediately repeated DNA. DNA sequence composition of the ancient tetraploid genomes suggests that the species derived from diploid ancestors of small genome size. 3. The average base composition and the base compositional heterogeneity are similar in all the species examined.

Animals↗

PubMed1986

Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments.

We have developed a novel efficient method, carrier-facilitated insertion, to insert small (150-600 bp) DNA fragments into plasmid vectors. This method employs a carrier segment of vector DNA to circumvent the difficulties in ligating two fragments together to generate a recombinant circle efficiently. We have used carrier-facilitated insertion to construct three genomic libraries of random (DNase I-generated) fragments from the Saccharomyces cerevisiae genome. One of these was an expression library, and the other two were promoter-cloning libraries. 87-90% of the Escherichia coli colonies in each library contained recombinant plasmids, and less than 3% of the recombinants contained more than one insert. Detection of open reading frames among the inserts in the expression library was accomplished by testing for beta-galactosidase activity. This methodology, unencumbered by the intrinsic disproportionality of cDNA libraries, can be used to identify and clone DNA that codes for a specific antigenic determinant. When used in combination with a method to detect and isolate random constitutive, repressible and inducible yeast promoters, these libraries should permit a comprehensive analysis of the yeast genome and its expression.

Cloning, Molecular↗

PubMed2006

Comparative genomic hybridization array analysis and real time PCR reveals genomic alterations in squamous cell carcinomas of the lung.

Genomic alterations have been identified in lung cancer tissues and reported in numerous studies. To analyze genomic aberrations in lung cancer patients, we used array comparative genomic hybridization (array CGH) in 14 squamous cell lung carcinoma (SqC) tissues. Copy number gain and loss in chromosomal regions were detected, and the corresponding genes were confirmed by real time PCR. Several frequently altered loci, including gain of 3q (36% of samples), were found. The most frequently identified losses were found at 14q32.33 (21% of samples). The relative degree of chromosomal change was analyzed using log2 ratios. High-level DNA amplifications (>0.8 log2 ratio) were detected at 20 regions in 1p, 2q, 3q, 4q, 6q, 7p, 8q, 9p, 10q, 12q, 14q and 19p. We found that the fold change levels were highest at EVI1 (3q26.2), LPP (3q27-28) and FHF-1 (3q28) gene loci. Our results show that array CGH is a useful tool for identification of gene alteration in lung cancer, and that the above-mentioned genes might represent potential candidate genes for pathogenesis and diagnosis of lung cancer.

Aged↗

PubMed1988

Sequence analysis of the 3'-end of the feline coronavirus FIPV 79-1146 genome: comparison with the genome of porcine coronavirus TGEV reveals large insertions.

The genetic information, carried on mRNA 6 of feline infectious peritonitis virus (FIPV) strain 79-1146, was determined by sequence analysis of cDNA clones derived from the 3' end of the FIPV genome. Two ORFs were found, encoding polypeptides of 11K (ORF-1) and 22K (ORF-2). The FIPV sequence was compared to the 3' end sequence of transmissible gastroenteritis virus (TGEV). ORF-1 has a homologous counterpart (ORF-X3) in the TGEV genome; both ORFs are located at the same position relative to the nucleocapsid gene. However, as a result of an in-frame insertion or deletion, ORF-1 is 69 nucleotides larger than ORF-X3. A similar event has occurred immediately downstream of ORF1: a 624-nucleotide segment, containing the complete ORF-2, is absent in the TGEV sequence. Most sequence similarity (98.5%) was found in the 3' noncoding sequences. ORF-X3 and ORF-1 are preceded by the sequence AACTAAAC, which is assumed to be the transcription-initiation signal in FIPV and TGEV (P.A. Kapke and D.A. Brian (1986) Virology 151, 41-49). By S1 nuclease analysis, the 5' end of FIPV RNA 6 was mapped immediately upstream of this sequence. A 700-nucleotide TGEV-specific RNA was found by cross-hybridization with an FIPV 3' end probe, suggesting that TGEV ORF-X3 is also carried on a separate mRNA. The differences at the 3' ends of the FIPV and TGEV genomes may be the result of RNA recombination events.

Amino Acid Sequence↗

PubMed2003

Intratumoral genomic heterogeneity in advanced head and neck cancer detected by comparative genomic hybridization.

Little is known about the extent of intratumoral genetic heterogeneity in head and neck squamous cell carcinoma (HNSCC). We therefore examined 79 stage III and IV primary HNSCCs (P) and matched lymph node metastases (M) for over- and underrepresentation of specific chromosome regions by comparative genomic hybridization (CGH). The overall ratio of gains and losses was higher in metastases than in primary tumors (4/1 vs. 2.5/1). Gains of 3q (78.1% P vs. 87.5% M) and 11q (78.1% P vs. 62.5% M) and deletions of 3p (43.8% P vs. 34.4% M) and 9p (31.3% P vs. 15.6% M) were most frequently detected. The highest rate of intratumoral discordance was observed for primary tumors and corresponding metastases (32.8%) compared with matched pairs of two metastases (26.5%) and of two anatomically distinct sides of one primary tumor (24.3%). Furthermore, the discordance rate was dependent on the primary tumor site (oral cavity 49.2%, oropharynx 31%, hypopharynx 30.3%, and larynx 27.3%). In some tumors, the extent of genomic discordance argues against a monoclonal origin. In conclusion, we found a high individual variation of intratumoral genomic heterogeneity depending on the localization and selection of matched pairs. These findings are of specific importance in view of establishing prognostic markers.

Aged↗

PubMed1997

Genome screening by comparative genomic hybridization.

Comparative genomic hybridization (CGH) provides a molecular cytogenetic approach for genome-wide scanning of differences in DNA sequence copy number. The technique is now attracting wide-spread interest, especially among cancer researchers. The rapidly expanding database of CGH publications already covers about 1500 tumors and is beginning to reveal genetic abnormalities that are characteristic of certain tumor types or stages of tumor progression. Six novel gene amplifications, as well as a locus for a cancer-predisposition syndrome, have been discovered based on CGH data. CGH has now been established as a first-line screening technique for cancer researchers and will serve as a basis for ongoing efforts to develop high-resolution next-generation genome scanning, such as the microarray technology.

Animals↗

PubMed1997

Sensitive and reliable detection of genomic imbalances in human neuroblastomas using comparative genomic hybridisation analysis.

Deletions of the short arm of chromosome 1, extra copies of chromosome 17q and MYCN amplification are the most frequently encountered genetic changes in neuroblastomas. Standard techniques for detection of one or more of these genetic changes are karyotyping, FISH analysis and LOH analysis by Southern blot or PCR. Each of these techniques has its own particular limitations. More recently, comparative genomic hybridisation (CGH) was introduced for detection of genomic imbalances including deletions, duplications and gene amplification. We evaluated the sensitivity and reliability of CGH for detection of the most frequently encountered genetic changes in neuroblastoma. For this purpose a panel of well-characterised neuroblastoma cell lines as well as a series of 11 primary neuroblastomas was analysed. Our results show that CGH is a valuable tool for the genetic characterisation of neuroblastomas, both for the detection of frequently occurring genomic imbalances and for the identification of previously unnoticed genetic changes.

Child, Preschool↗

PubMed1998

Oral squamous cell carcinomas are characterized by a rather uniform pattern of genomic imbalances detected by comparative genomic hybridisation.

Total genomic DNA sampled from 20 oral squamous cell carcinomas (SCCs) and from four SCC cell lines, was examined for genomic imbalances using comparative genomic hybridisation (CGH). Gains and losses of DNA copy number aberrations (CNAs) were found in the primary tumours, but also in the cell lines at a varying number. The patterns of CNAs proved to be rather peculiar in oral SCCs, gains of genetic material clearly dominating compared with losses, and a rather high uniformity of these patterns was an impressive finding. Hypersomies of whole chromosomes, e.g. numbers 17 and 19 or of whole chromosome arms, e.g. 20q, were particularly evident. The segments most frequently gained in oral SCCs were 3q26-q27, 5p15 and 9q34 (16 of 20 tumours each), as well as 1p36.3, 8q24, 10q26, 19 and 20q (15/20 each). Among the 15 tumours with more than 10 CNAs, all showed these imbalances. 11q13 was a band often involved in increases (14/20 tumours), but in several tumours was involved in amplification of DNA copy number. Several other chromosomal segments over represented in more than 60% of the tumours, as, for example, 12q24, 15q22-q24, 16p13.2 and 17q (14/20 tumours each), 6q26-qter, 7p22, 12p12.2-p13, 14q31-q32.2 (13/20) and 1q32-q41, 2q37, 16q23-q24 (12/20 each). In contrast, loss of material affected only a few chromosomal segments, as, for example, 3p12 (12 of the 20 tumours), 5q21 (10/20), 6q13 (8/20). The peculiarities of these findings, in some respect, differ from those found in other epithelial tumours, suggesting a high impact of environmental factors in the generation and progression of these tumours.

Adult↗

PubMed2002

Finding genes for bipolar disorder in the functional genomics era: from convergent functional genomics to phenomics and back.

Psychiatric genetics, while promising to unravel the mechanisms of psychiatric disorders, has proven to be a challenging field. Psychiatric disorders, like other common genetic traits, are complex and heterogeneous. Psychiatric genetics has also suffered from a lack of quantifiable, biology-based phenotypes. However, the field is currently at an opportune moment. The work of various investigators is on the verge of paying rich dividends. Efforts at positional cloning are being greatly accelerated by the fruits of the Human Genome Project. New tools of functional genomics, such as expression profiling and proteomics, are being applied to animal models. These two methods can complement each other in an approach we have termed convergent functional genomics. Lastly, improvements in the measurement of biologically distinct endophenotypes--or phenomic--will lead to a better understanding of the mapping of genes to phenotypes in both animal and human systems.

Journal Article↗

PubMed2001

The genomics of land plant chloroplasts: Gene content and alteration of genomic information by RNA editing.

The entire nucleotide sequence of the chloroplast genome has been determined from 12 land plants. The gene content and arrangement are relatively uniform from species to species, and the genome contains an average of 111 identified gene species (except Epifagus). Chloroplast genes can be classified into three main categories: Genes for the photosynthetic apparatus, those for the transcription/translation system, and those related to biosyntheses. The genes encoding components of the photosynthesis apparatus have been identified by protein chemical analyses from higher plants, Chlamydomonas and cyanobacteria, and then by chloroplast transformation techniques using tobacco and Chlamydomonas. The genes for subunits of RNA polymerases and of ribosomes were initially deduced similarity to those in E. coli, and later confirmed by protein analyses. Coding information is often modified at the level of transcripts by RNA editing (mostly C-U changes), resulting in amino acid substitutions and creation of novel reading frames. Perspectives of chloroplast genomics are discussed.

Journal Article↗

PubMed2002

Mammary tumors in mice conditionally mutant for Brca1 exhibit gross genomic instability and centrosome amplification yet display a recurring distribution of genomic imbalances that is similar to human breast cancer.

BRCA1 mutation carriers have an increased susceptibility to breast and ovarian cancer. Excision of exon 11 of Brca1 in the mouse, using a conditional knockout (Cre-loxP) approach, results in mammary tumor formation after long latency. To characterize the genomic instability observed in these tumors, to establish a comparative map of chromosomal imbalances and to contribute to the validation of this mouse model of breast cancer, we have characterized chromosomal imbalances and aberrations using comparative genomic hybridization (CGH), and spectral karyotyping (SKY). We found that all tumors exhibit chromosome instability as evidenced by structural chromosomal aberrations and aneuploidy, yet they display a pattern of chromosomal gain and loss that is similar to the pattern in human breast carcinomas. Of note, nine of 15 tumors exhibited a gain of distal chromosome 11, a region that is orthologous to human chromosome 17q11-qter, the mapping position of Erbb2. However, our analysis suggests that genes distal to Erbb2 are the main targets of amplification. Four of the tumors also exhibited a copy number loss of proximal chromosome 11 (11A-B), a region orthologous to human 17p. In eight of the tumors we observed whole or partial gain of chromosome 15 centering on 15D2-D3 (orthologous to human chromosome 8q24), the map location of the c-Myc gene, and six of the tumors exhibited copy number loss of whole or partial chromosome 14, including 14D3, the map location of Rb1. We conclude that despite the tremendous shuffling of chromosomes during the course of mammalian evolution, the pattern of genomic imbalances is conserved between BRCA1-associated mammary gland tumors in mice and humans. Western blot analysis showed that while p53 is absent or mutated in some tumors, at least two tumors revealed wild-type protein, suggesting that other genetic events may lead to tumorigenesis. Similar to BRCA1-deficient mouse embryonic fibroblasts, the tumor cells contained supernumerary functional centrosomes with intact centrioles whose presence results in multipolar mitoses and aneuploidy.

Aneuploidy↗

PubMed1998

Genomic organization of the human mucin gene MUC5B. cDNA and genomic sequences upstream of the large central exon.

The complete structure of the DNA encoding the polypeptide chain of human mucin MUC5B has been determined. In this paper, we report the full-length cDNA (3886 bp) and genomic (15,143 bp) sequences upstream of the unusually large central exon of the human mucin gene MUC5B. This region, composed of 29 exons, encodes 1283 amino acid residues. Exon sizes vary from 44 to 262 bp, and intron sizes range from 87 to 1703 bp. We determined the 5'-end of MUC5B by performing rapid amplification of cDNA ends-polymerase chain reaction experiments leading to the same length of the amplified product and by using primer extension experiments. A putative translation start site was found at nucleotide +37. We compared the amino-terminal region of MUC5B with those of pro-von Willebrand Factor, MUC2 and MUC5AC, and animal mucins, RMuc2, PSM, and FIM-B.1. The primary amino acid sequence with a high content of cysteine residues demonstrates a high degree of similarity with other members of the 11p15 mucin gene family, particularly MUC5AC. The complete genomic organization and both full-length genomic and cDNA sequences of MUC5B have been elucidated. This gene contains 48 exons and encodes 5662 amino acid residues to give a polypeptide with a Mr approximately 600,000.

Amino Acid Sequence↗

PubMed2003

The complete nucleotide sequence of goat (Capra hircus) mitochondrial genome. Goat mitochondrial genome.

The goat mtDNA sequences reported to date are fragmentary. By using both in silico cloning procedure and conventional molecular biology techniques we have determined the complete nucleotide sequence of the goat (Capra hircus) mitochondrial genome. The length of the sequence was 16.640 bp. Genes responsible for 12S and 16S rRNAs, 22 tRNAs and 13 protein-coding regions are found. The genome organization is conformed to those of other mitochondrial genomes. Comparison between the 13 protein coding genes of goat, cow and sheep reveals that the difference range from 1.2 to 12.2% with a mean of 7.3% between goat and cow and from 0 to 15.6% (mean 4.7%) between goat and sheep.

Animals↗