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PubMed1989

The class II genes of the rat MHC.

Genes that encode class II Ag from the MHC of the rat, the RT1 region, have been isolated as a series of cosmid clones. The cosmids define two clusters, each of which contains three identifiable sequences; one homologous to alpha-chain and two to beta-chain genes. Both the serologically identified rat class II Ag have been expressed in mouse L cell fibroblasts after the introduction of each alpha-chain gene along with a beta-chain gene from the same cluster. There are substantial homologies to the I region of the mouse H-2 complex in the presence, location, orientation, and expression of the six identified sequences from the rat RT1, supporting the view that the overall organization of the two gene complexes has remained conserved since the species separated.

Animals↗
PubMed1988

Molecular cloning of a novel oncogene generated by DNA recombination during transfection.

Genomic DNA of a Ewing's sarcoma cell line and pKOneo were cotransfected into NIH3T3 cells and assayed for tumorigenicity in nude mice. Primary transfected tumors all contained human DNA. One transfected sequence was retained through secondary and tertiary tumors, and was cloned in four overlapping cosmids covering 51.5-kb human DNA flanked on both sides with mouse DNA. The cloned sequence, though continuous in transfected tumors, originated from three major genetic elements discontiguous in human cells and recombined during transfection. Accordingly there was one species of hybrid RNA transcripts of 9 kb in the transfected tumors and no transcripts in human cells, including Ewing's sarcoma cells. Evidence that the cloned sequence is a biologically active novel oncogene was provided by transfection of a mixture of two cosmid clones which generated tumors where they were transcribed into 9-kb RNA. The oncogene was named tre to designate its origin from transfection recombined DNA molecules.

Animals↗
PubMedDate not supplied

[Vectors--derivatives of phage lambda for construction and analysis of genome libraries].

Basic features of lambda phage derived, cosmid and plasmid vectors are described. Plasmid vectors combine the most useful features of phage and plasmid vectors. Plasmids can exist in vivo as a plasmid or as a phage. Plasmid vectors are similar to large capacity phage vectors, but can be maintained in vivo without a stuffer fragment. That is why plasmids are easier in preparation for cloning than phage vectors. The yield of recombinants is higher with plasmid vectors (up to 3.10(6)) and the background of non-recombinants in the library is lower. Analysis of recombinant plasmids is more simple and effective than analysis of recombinant phages and plasmids. Probably plasmid vectors will soon be widely used instead of phage or cosmid vectors for genomic libraries construction and analysis.

Bacteriophage lambda↗
PubMed1995

An integrated high-resolution physical map of the DPC/BRCA2 region at chromosome 13q12.

We identified a homozygous deletion in a pancreatic carcinoma (DPC) that localized to a 1-cM region at chromosome 13q12.3, which lay within the 6-cM locus of familial breast cancer susceptibility (BRCA-2). Here we present a physical map of the region, consisting of YAC, PAC, and cosmid contigs. The YAC contig comprises 16 clones that together span the entire BRCA2 region. The PAC contig comprises 22 clones that together span the DPC region. Seventy cosmid clones were localized within and near the DPC region. Thirty-five sequence-tagged sites were defined and localized within the map. The map indicates the size of the DPC region to be near 250 kb, and provides mapped and cloned resources for the search for the putative tumor suppressor gene(s) in the region.

Base Sequence↗
PubMed1994

Genetic and physical mapping of human recoverin: a gene expressed in retinal photoreceptors.

PURPOSE: Recoverin is a calcium-binding protein that may be involved in phototransduction in mammalian retinal photoreceptors, and is considered to be a candidate gene for retinitis pigmentosa. This study was undertaken to develop the recoverin locus into a polymorphic marker for future linkage studies on retinitis pigmentosa families. METHODS: A human genomic cosmid clone was isolated and used to map the recoverin gene to a human chromosome through hybridization to a panel of somatic hybrid cell line DNAs, and to human metaphase chromosomes by fluorescence in situ hybridization. A dinucleotide repeat polymorphism located within the coding region of the recoverin gene was identified, and used to genetically map the recoverin gene relative to index markers. In addition, three restriction fragment length polymorphisms revealed by the cosmid clone were identified and characterized. RESULTS: Hybridization to the somatic hybrid cell line DNAs localized the recoverin gene to chromosome 17. Recoverin was further localized to 17p12-p13 by fluorescence in situ hybridization. The dinucleotide repeat polymorphism and restriction fragment length polymorphisms at the recoverin locus have a cumulative polymorphic information content = 0.71. CONCLUSIONS: These polymorphic markers and additional closely linked markers will be useful for linkage analysis of families with retinitis pigmentosa.

Base Sequence↗
PubMed1993

Structural analysis of the human ret proto-oncogene using exon trapping.

A genomic contig of the human ret protooncogene was created with four overlapping cosmid clones isolated from two libraries. After southern analysis with portions of the ret cDNA, eight cosmid fragments were analysed in detail for the presence of ret exons using exon trapping. PCR products corresponding to spliced exons were isolated and subcloned. Exon boundaries were delineated by comparison of the PCR product sequence and the published ret cDNA sequence. The exons were initially positioned on a genomic map defined by BamHI, EcoRI and HindIII restriction sites. The positions of the exons were then refined by amplifying genomic DNA using primer pairs derived from one or more exons along the ret gene, the length of the PCR product indicating the approximate genomic distance between the exon sequences. The ret proto-oncogene is composed of at least 20 exons, ranging in size from 60 bp to 287 bp, distributed along 30 kb of genomic DNA. The extracellular domain is encoded by 10 exons and the cytoplasmic domain by 9 exons. The transmembrane domain is encoded by a single exon.

Base Sequence↗
PubMed1993

Detection of a molecular deletion at the DXS732 locus in a patient with X-linked hypohidrotic ectodermal dysplasia (EDA), with the identification of a unique junctional fragment.

X-linked hypohidrotic ectodermal dysplasia (EDA) has been localized to the Xq12-q13.1 region. A panel of genomic DNA samples from 80 unrelated males with EDA has been screened for deletions at seven genetic loci within the Xq12-13 region. A single individual was identified with a deletion at the DXS732 locus by hybridization with the mouse genomic probe pcos169E/4. This highly conserved DNA probe is from locus DXCrc169, which is tightly linked to the Ta locus, the putative mouse homologue of EDA. The proband had the classical phenotype of EDA, with no other phenotypic abnormalities, and a normal cytogenetic analysis. A human genomic DNA clone, homologous to pcos169E/4, was isolated from a human X-chromosome cosmid library. On hybridization with the cosmid, the proband was found to be only partially deleted at the DXS732 locus, with a unique junctional fragment identified in the proband and in three of his maternal relatives. This is the first determination of carrier status for EDA in females, by direct mutation analysis. Failure to detect deletion of the other loci tested in the proband suggests that the DXS732 locus is the closest known locus to the EDA gene. Since the DXS732 locus contains a highly conserved sequence, it must be considered to be a candidate locus for the EDA gene itself.

Adult↗
PubMed1993

Narrowing the position of the Treacher Collins syndrome locus to a small interval between three new microsatellite markers at 5q32-33.1.

Treacher Collins syndrome (TCOF1) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCOF1 locus has been localized to chromosome 5q32-33.2. In the present study we have used the combined techniques of genetic linkage analysis and fluorescence in situ hybridization (FISH) to more accurately define the TCOF1 critical region. Cosmids IG90 and SPARC, which map to distal 5q, encompass two and one hypervariable microsatellite markers, respectively. The heterozygosity values of these three markers range from .72 to .81. Twenty-two unrelated TCOF1 families have been analyzed for linkage to these markers. There is strong evidence demonstrating linkage to all three markers, the strongest support for positive linkage being provided by haplotyping those markers at the locus encompassed by the cosmid IG90 (Zmax = 19.65; theta = .010). FISH to metaphase chromosomes and interphase nuclei established that IG90 lies centromeric to SPARC. This information combined with the data generated by genetic linkage analysis demonstrated that the TCOF1 locus is closely flanked proximally by IG90 and distally by SPARC.

Base Sequence↗
PubMed1995

Expression of the cbbLcbbS and cbbM genes and distinct organization of the cbb Calvin cycle structural genes of Rhodobacter capsulatus.

Rhodobacter capsulatus fixes CO2 via the Calvin reductive pentose phosphate pathway and, like some other nonsulfur purple bacteria, is known to synthesize two distinct structural forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Cosmid clones that hybridized to form I (cbbLcbbS) and form II (cbbM) RubisCO gene probes were isolated from a genomic library of R. capsulatus strain SB1003. Southern blotting and hybridization analysis with gene-specific probes derived from Rhodobacter sphaeroides revealed that R. capsulatus cbbM is clustered with genes encoding other enzymes of the Calvin cycle, including fructose 1,6/sedoheptulose 1,7-bisphosphatase (cbbF), phosphoribulokinase (cbbP), transketolase (cbbT), glyceraldehyde-3-phosphate dehydrogenase (cbbG), and fructose 1,6-bisphosphate aldolase (cbbA), as well as a gene (cbbR) encoding a divergently transcribed LysR-type regulatory protein. Surprisingly, a cosmid clone containing the R. capsulatus form I RubisCO genes (cbbL and cbbS) failed to hybridize to the other cbb structural gene probes, unlike the situation with the closely related organism R. sphaeroides. The form I and form II RubisCO genes were cloned into pUC-derived vectors and were expressed in Escherichia coli to yield active recombinant enzyme in each case. Complementation of a RubisCO-deletion strain of R. sphaeroides to photosynthetic growth by R. capsulatus cbbLcbbS or cbbM was achieved using the broad host-range vector, pRK415, and R. sphaeroides expression vector pRPS-1.

Chromosome Mapping↗
PubMedDate not supplied

Characterisation of X;17(q12;p13) translocation breakpoints in a female patient with hypomelanosis of Ito and choroid plexus papilloma.

An X;17 translocation breakpoint was characterised in a 5-year-old female with hypomelanosis of Ito (HI) who exhibits characteristic hypopigmented lesions, psychomotor retardation, and choroid plexus papilloma. A YAC clone containing the locus DXS1 from Xq12 was found by fluorescence in situ hybridisation to cross the translocation breakpoint. Cosmid clones positive for DXS1 were used to identify and clone the translocation junction fragment from the patient's DNA. A chromosome-17-specific DNA fragment was isolated and used to identify cosmid clones crossing the translocation from chromosome 17p13. Exon trapping identified two known genes from chromosome 17: FMR1L2 (the fragile X mental retardation syndrome like protein 2) and SHBG (human sex hormone-binding globulin). Mapping the FMR1L2 and SHBG genes showed that neither gene was disrupted by the translocation.

Brain Neoplasms↗
PubMed1997

Cloning and characterization of the regulatory genes phlR1 and phlR2 involved in phenol metabolism from Alcaligenes eutrophus JMP134.

One mutant (AEK201) of Alcaligenes eutrophus JMP134 deficient in phenol metabolism was isolated by transposon mutagenesis using pSUP2021, a suicide plasmid. The 14.5 kb EcoRI fragment containing Tn5 and flanking DNA was cloned from AEK201 and used to probe a gene bank of wild type by colony hybridization. All five positive cosmids isolated rendered AEK201 to grow on phenol. The data from subcloning revealed that a trans-acting factor encoded on the 2.3 kb SalI-HindIII fragment, which is common to all cosmids, allowed the mutant to restore three enzyme activities tested (phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase). This fragment seems to act as a positive regulator on the entire phenol pathway. Another regulatory segment was subcloned from the 16.8 kb HindIII fragment on which phenol hydroxylase and catechol 2,3-dioxygenase activities were carried [Kim, Y., Ayoubi, P., and Harker, A. R. (1996) Appl. Environ. Microbiol. 62, 3227-3233]. The expression of phenol hydroxylase activity was entirely repressed in the presence of this segment in Pseudomonas aeruginosa PAO1c, but the enzyme activity was increased in A. eutrophus AEK301, suggesting that this trans-acting factor is both an activator and a repressor for phenol hydroxylase. Possible regulatory mechanisms for the phenol pathway in A. eutrophus JMP134 are discussed.

Alcaligenes↗
PubMed1997

Designing of plant artificial chromosome (PAC) by using the Chlorella smallest chromosome as a model system.

As a model for plant-type chromosomes, we have been characterizing molecular organization of the Chlorella vulgaris C-169 chromosome I. To identify chromosome structural elements including the centromeric region and replication origins, we constructed a chromosome I specific cosmid library and aligned each cosmid clones to generate contigs. So far, more than 80% of the entire chromosome I has been covered. A complete clonal physical reconstitution of chromosome I provides information on the structure and genomic organization of plant genome. We propose our strategy to construct an artificial chromosome by assembling the functional chromosome structural elements identified on Chrorella chromosome I.

Centromere↗
PubMed1998

Submicroscopic deletion of chromosome 16p13.3 in patients with Rubinstein-Taybi syndrome.

The Rubinstein-Taybi syndrome (RTS) is a well-defined entity characterized by growth and mental retardation, broad thumbs and halluces, and typical face. The RTS locus was assigned to 16p13.3, and interstitial submicroscopic deletions of this region (RT1 cosmid, D16S237) were initially identified in 25% of RTS patients. The gene for the human CREB binding protein, the transcriptional coactivator CBP, is included in the RT1 cosmid, and mutations in CBP have recently been identified in nondeleted RTS patients. We investigated 30 French patients with RTS. Among these patients, 3 had the RT1 microdeletion (frequency 10%). There is no obvious phenotypic difference between the patients with and without the RT1 deletion. The RT1 probe appears useful for confirmation of the diagnosis but is of little interest as a screening tool. By pooling data including the previous series and our current series, the cumulative frequency of the 16p13.3 microdeletion is 11.9% (19 in 159). This frequency of approximately 12% deleted patients appears more accurate than the 25% previously reported. Molecular investigations of CBP are in process in our series to clarify the cause of RTS.

CREB-Binding Protein↗
PubMed1996

Interphase fluorescence in situ hybridization detection of t(2;13)(q35;q14) in alveolar rhabdomyosarcoma--a diagnostic tool in minimally invasive biopsies.

The identification of t(2;13)(q35;q14) is a useful aid in the accurate diagnosis of rhabdomyosarcoma, distinguishing it from other small round cell tumours and supporting the distinction between alveolar and embryonal forms. Cytogenetic analysis is difficult and with the increased use of minimally invasive biopsy methods and primary chemotherapy, adequate tumour material is not always available. To overcome these difficulties, two-colour interphase fluorescence in situ hybridization (FISH) to detect t(2;13)(q35;q14) was developed and its utility in assessing minimally invasive biopsies was investigated. Two cosmid clones which mapped proximal or distal to the breakpoint region 13q14 and one yeast artificial chromosome clone that mapped distal to the 2q35 breakpoint were identified. In interphase cells containing t(2;13)(q35;q14), the configuration of cosmid and yeast artificial chromosome signals demonstrated the presence of the translocation. Five cases of rhabdomyosarcoma were analysed by interphase FISH. The t(2;13)(q35;q14) was detected in all four alveolar tumours and was confirmed by cytogenetics in two cases, but was absent in one embryonal tumour. This sensitive detection method is applicable to minimal amounts of fresh or frozen tumour.

Adolescent↗
PubMed1996

Loss of chromosome 11 and 11 p/q imbalances in bladder cancer detected by fluorescence in situ hybridization.

To identify chromosomal imbalances in non-diploid transitional-cell carcinoma (TCC) of the bladder we performed double-target in situ hybridization (FISH), using the centromeric probe for chromosome 11 together with 2 cosmid probes located on the 11p and 11q arm in the proximity of the telomere. The FISH protocol was optimized to ensure a highly efficient and reproducible detectability of all 3 targets. As a consequence, it was possible to calculate ratios between the number of spots obtained with cosmid and centromere probes. Furthermore, the number of chromosomes 11 present was compared with the DNA index and the chromosome ploidy as obtained with other chromosome centromere probes. In this study we found that: (i) in 54 diploid TCCs a monosomy for chromosome 11 was detected in only one case; (ii) chromosome 11 was completely lost in 9 of 16 non-diploid TCCs; (iii) in 8 of these 16 non-diploid tumors an imbalance was observed between the 11p and 11q arm, in 4 of these cases a complete loss of chromosome 11 being observed in addition; (iv) the copy number counted for 11q was always identical to the 11 centromere number, except in one case, indicating a loss of 11p in the cases with imbalances. In total, 13 of 16 non-diploid TCCs (81%) showed either a loss of a complete chromosome 11, of (part of) the 11p arm, or both. Therefore we concluded that during tetra- or aneuploidization in TCCs, (part of) chromosome 11 is lost. In addition, our results indicate that under-representation of chromosome 11p occurs in the majority of the tumor cells, supporting the idea that loss of these sequences is an important step in the development of TCC.

Chromosomes, Human, Pair 11↗
PubMed1998

Identification of a 100-kb region of common allelic loss on chromosome bands 10q25-q26 in human endometrial cancer.

In human endometrial cancer, we have previously identified a 790-kb region of common allelic loss in chromosome bands 10q25-q26, flanked by D10S587 and D10S1723. We constructed a contig covering the entire deleted region using YACs, PACs, and BACs. Five overlapping cosmid clones derived from YAC clones completely covered the entire deleted region: its size was estimated to be no larger than 200 kb. We further performed two-color fluorescence in situ hybridization (FISH) analysis to confirm the deletion and narrowed down the deleted region to 100 kb or less; it was covered by three overlapping cosmid clones that were included in one BAC clone. Restriction endonuclease mapping identified a region in which NotI, SalI, SmaI, and Xhol were clustered, suggesting the possible existence of a CpG island.

Chromosome Banding↗
PubMed1999

Mapping of the breakpoints on the short arm of chromosome 17 in neoplasms with an i(17q).

Isochromosomes are monocentric or dicentric chromosomes with homologous arms that are attached in a reverse configuration as mirror images. With an incidence of 3-4%, the i(17q) represents the most frequent isochromosome in human cancer. It is found in a variety of tumors, particularly in blast crisis of chronic myeloid leukemia (CML-BC), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), and medulloblastoma (MB), and indicates a poor prognosis. To determine the breakpoints on the molecular genetic level, we analyzed 18 neoplasms (six CML, four AML, one NHL, and seven MB) with an i(17q) and two MB with a pure del(17p) applying fluorescence in situ hybridization (FISH) with yeast artificial chromosome (YAC) clones, P1-artificial chromosome (PAC) clones, and cosmids from a well-characterized contig covering more than 6 Mb of genomic DNA. We identified four different breakpoint cluster regions. One is located close to or within the centromere of chromosome 17 and a second in the Charcot-Marie-Tooth (CMT1A) region at 17(p11.2). A third breakpoint was found telomeric to the CMT1A region. The fourth, most common breakpoint was detected in MB, AML, and in CML-BC specimens and was bordered by two adjacent cosmid clones (clones D14149 and M0140) within the Smith-Magenis syndrome (SMS) region. These results indicate that the low copy number repeat gene clusters which are present in the CMT and SMS regions may be one of the factors for the increased instability that may trigger the formation of an i(17q).

Adolescent↗
PubMed1995

FISH analysis in Prader-Willi and Angelman syndrome patients.

We report on a combined high resolution cytogenetic and fluorescent in situ hybridization study (FISH) on 15 Prader-Willi syndrome (PWS) and 14 Angelman syndrome (AS) patients. High resolution banding showed a microdeletion in the 15q11-q13 region in 7 out of 15 PWS patients, and FISH analysis of the D15S11 and SNRPN cosmids demonstrated absence of the critical region in three additional cases. Likewise 8 out of 14 AS patients were found to be deleted with FISH, using the GABRB3 specific cosmid, whereas only 4 of them had a cytogenetically detectable deletion.

Angelman Syndrome↗