Microisolation of the chicken Z chromosome and construction off microclone libraries

Genome ◽  
1997 ◽  
Vol 40 (6) ◽  
pp. 865-872 ◽  
Author(s):  
Régis Zimmer ◽  
Alon Haberfeld ◽  
Ann M. Verrinder Gibbins
Keyword(s):  
Light Microscope ◽  
Genomic Dna ◽  
Size Range ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Z Chromosome ◽  
Mitotic Metaphase ◽  
Simple Method ◽  
Dna Library ◽  
Male Chicken

A simple method was used to adapt a standard light microscope for the collection of chicken Z chromosomes from mitotic-metaphase spreads. The DNA of the collected chromosomes was enzymatically amplified using a partially degenerate primer. The resulting sequences, within a size range of 200–800 bp, were cloned to produce a Z chromosome DNA library, using blunt-end ligation into a SmaI-digested pUC18 plasmid (the SureClone system; Pharmacia, U.S.A.). The microcloning experiments produced 1250 clones; the size range of the cloned inserts was 250–800 bp, with an average of 480 bp (176 clones examined). Using male chicken genomic DNA as a probe, 10 out of 17 randomly selected clones showed strong positive signals on Southern blots, confirming the origin of the inserts as chicken DNA. In addition, the Z-chromosome origin of a selected microclone was verified in a semiquantitative Southern blot hybridization that showed positive signals with intensities that were approximately twice as strong for male (ZZ) as for female (ZW) chicken genomic DNA when the clone was used as a probe. The value of these libraries in further analysis of the chicken Z chromosome is discussed.Key words: microdissection, microcloning, chicken Z chromosome.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome

1986 ◽  
Vol 6 (12) ◽  
pp. 4516-4525
Author(s):  
K G Coleman ◽  
H Y Steensma ◽  
D B Kaback ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Cloning ◽  
Genomic Dna ◽  
Shuttle Vector ◽  
Blot Hybridization ◽  
Yeast Genome ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library ◽  
Chromosome I

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae

Genome ◽  
2002 ◽  
Vol 45 (4) ◽  
pp. 777-783 ◽  
Author(s):  
Masahiro Hizume ◽  
Fukashi Shibata ◽  
Ayako Matsumoto ◽  
Yukie Maruyama ◽  
Eiji Hayashi ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Repetitive Dna ◽  
Genomic Dna ◽  
Repeat Unit ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Repeat Sequence ◽  
Proximal Region

Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0–3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.Key words: AT-rich tandem repetitive DNA, fluorescence in situ hybridization, Larix, proximal DAPI band.

Centromeric tandem repeat from the chaffinch genome: Isolation and molecular characterization

Genome ◽  
2001 ◽  
Vol 44 (1) ◽  
pp. 96-103 ◽  
Author(s):  
A F Saifitdinova ◽  
S E Derjusheva ◽  
A G Malykh ◽  
V G Zhurov ◽  
T F Andreeva ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Blot Hybridization ◽  
Restriction Enzymes ◽  
Southern Blot Hybridization ◽  
Repeated Sequence ◽  
Centromeric Heterochromatin ◽  
Specific Primers ◽  
Fringilla Coelebs ◽  
New Family

A new family of avian centromeric satellites is described. The highly repeated sequence, designated FCP (Fringilla coelebs PstI element), was cloned from the 500-bp PstI digest fraction of the chaffinch (Fringilla coelebs L.) genomic DNA, sequenced, and characterized. The FCP repeat was found to have 505–506 bp length of monomer, 57% content of GC, to compose about 0.9% of the chaffinch genome, and to be highly methylated. Results of Southern-blot hybridization of cloned FCP element onto genomic DNA digested with different restriction enzymes, and sequencing directly from total genomic DNA using FCP-specific primers and ThermoFidelase enzyme (Fidelity Systems Inc.) were in agreement with a tandem arrangement of this repeat in the chaffinch genome. Five positions of single-nucleotide polymorphism (SNP) were found in the FCP monomers using direct genomic sequencing. Fluorescence in situ hybridization (FISH) with FCP probe and primed in situ labelling (PRINS) with FCP specific primers showed that the FCP elements occupy pericentric regions of all chaffinch chromosomes. On chromosome spreads, the fluorescent signals were also observed in the intercentromeric connectives between nonhomologous chromosomes. The results suggest that the centromeric FCP repeat is responsible for chromosome ordering during mitosis in chaffinch.Key words: satellite DNA, centromeric heterochromatin, Fringilla coelebs.

Characterization of the Opisthorchis viverrini genome

1991 ◽  
Vol 65 (1) ◽  
pp. 51-54 ◽  
Author(s):  
R. Sermswan ◽  
S. Mongkolsuk ◽  
S. Sirisinha
Keyword(s):  
Southern Blot ◽  
Genomic Dna ◽  
Blot Hybridization ◽  
Restriction Enzymes ◽  
Southern Blot Hybridization ◽  
Fasciola Gigantica ◽  
Opisthorchis Viverrini ◽  
Repeated Dna ◽  
Dna Elements

ABSTRACTThe methylations of trematode genomic DNA were analyzed using restriction enzymes and Southern blot hybridization. Restriction enzymes MspI, HpaII, HhaI were used to probe CpG methylation while MboI, Sau3A, DpnI were used for A methylation. The results revealed that Opisthorchis viverrini, Fasciola gigantica and Gigantocotyle siamensis had neither CpG nor A methylations. The presence of highly repeated DNA elements was also demonstrated in O. viverrini genomic DNA.

CHARACTERIZATION OF THE GENE FOR HUMAN HISTIDINE-RICH GLYCOPROTEIN

1987 ◽  
Author(s):  
T Koide
Keyword(s):  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Single Chain ◽  
Structural Domains ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Human Genomic ◽  
Considerable Homology ◽  
Amino Acid Segment

Human histidine-rich glycoprotein (HRG) is a single-chain glycoprotein in plasma which is considered to modulate a coagulation and fibrinolysis system with the ability to bind to heparin, plasminogen, fibrinogen, thrombospondin, etc. Recently we have elucidated the primary structure of HRG by determining the nucleotide sequence of its cDNA, and showed that HRG is composed of several different types of internal repeats, each one of which shows considerable homology with the functional and/or structural domains of other proteins including high molecular weight kininogen, antithrombin III, cystatins, and proline-rich protein and peptide. Thus, the multifunctional property of HRG was suggested to be due to its multi-domain structure. In the present studies, a human genomic DNA library, cloned in the bacteriophage vector Charon 4A, was screened for HRG gene using a full-length cDNA coding for human IMI as a probe. A total of 7 clones were isolated from 6 × 105 phage and each was plaque purified. The entire HRG gene is represented in 3 genomic inserts with overlapping sequences that carry human DNA spanning 30 kb. Overlapping gene fragments were subcloned into pUC9 and characterized by Southern blot hybridization using 5’ and 3’ end probes isolated from human HRG cDNA and by DNA sequencing. These studies have shown that the gene for human HRG spans about 9 kb and consists of at least 5 exons and 4 introns. The putative histidine-rich region consisted of 12 tandemly repeated sequences of a 5 amino acid segment and 2 proline-rich regions contiguous to it are likely to be involved within one exon.

scholarly journals Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

1986 ◽  
Vol 6 (12) ◽  
pp. 4516-4525 ◽  
Author(s):  
K G Coleman ◽  
H Y Steensma ◽  
D B Kaback ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Cloning ◽  
Genomic Dna ◽  
Shuttle Vector ◽  
Blot Hybridization ◽  
Yeast Genome ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library ◽  
Chromosome I

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

scholarly journals Methyl-CpG-Binding PCR of Bloodspots for Confirmation of Fragile X Syndrome in Males

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Ching-Cherng Tzeng ◽  
Chiou-Ping Liou ◽  
Chien-Feng Li ◽  
Ming-Chi Lai ◽  
Li-Ping Tsai ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Promoter Methylation ◽  
Fragile X ◽  
Blot Hybridization ◽  
Methylation Status ◽  
Southern Blot Hybridization ◽  
Detection Sensitivity ◽  
Simple Method ◽  
X Chromosomes ◽  
Blinded Manner

This study demonstrates that methyl-CpG-binding PCR (MB-PCR) is a rapid and simple method for detecting fragile X syndrome (FXS) in males, which is performed by verifying the methylation status of theFMR1promoter in bloodspots. Proteins containing methyl-CpG-binding (MB) domains can be freeze-stored and used as stocks, and the entire test requires only a few hours. The minimum amount of DNA required for the test is 0.5 ng. At this amount, detection sensitivity is not hampered, even mixing with excess unmethylated alleles up to 320 folds. We examined bloodspots from 100 males, including 24 with FXS, in a blinded manner. The results revealed that the ability of MB-PCR to detectFMR1promoter methylation was the same as that of Southern blot hybridization. Since individuals with 2 or more X chromosomes generally have methylatedFMR1alleles, MB-PCR cannot be used to detect FXS in females.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 967-978 ◽  
Author(s):  
C. Viana-Niero ◽  
P. E. de Haas ◽  
D. van Soolingen ◽  
S. C. Leão
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phospholipase C ◽  
Genetic Polymorphisms ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Clinical Isolates ◽  
Significant Similarity ◽  
Genomic Deletions ◽  
Genes Encoding ◽  
Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

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