Microdissection of and microcloning from the short arm of human chromosome 2

1986 ◽  
Vol 6 (11) ◽  
pp. 3826-3830
Author(s):  
G P Bates ◽  
B J Wainwright ◽  
R Williamson ◽  
S D Brown
Keyword(s):  
Human Chromosome ◽  
Dna Sequences ◽  
Chromosomal Location ◽  
Genetic Diseases ◽  
Repetitive Sequences ◽  
Single Copy ◽  
Chromosome 2 ◽  
Insert Size ◽  
Base Pairs ◽  
The Mean

A bank of cloned DNA sequences from the distal half of the short arm of human chromosome 2 was generated by using microdissection and microcloning techniques. DNA was purified from 106 chromosomal fragments, manually dissected from peripheral lymphocytes in metaphase, and cloned into the EcoRI site of lambda gt10. A total of 257 putative recombinants were recovered, of which 41% were found to contain human inserts. The mean insert size was 380 base pairs (median size, 83 base pairs), and fewer than 10% of the clones contained highly repetitive sequences. All single-copy sequences examined were shown to map to the short arm of chromosome 2 by using hybrid panels. This technique provides a rapid method of isolating probes specific to a human subchromosomal region to generate linked markers to genetic diseases for which the chromosomal location is known.

scholarly journals Microdissection of and microcloning from the short arm of human chromosome 2.

1986 ◽  
Vol 6 (11) ◽  
pp. 3826-3830 ◽  
Author(s):  
G P Bates ◽  
B J Wainwright ◽  
R Williamson ◽  
S D Brown
Keyword(s):  
Human Chromosome ◽  
Dna Sequences ◽  
Chromosomal Location ◽  
Genetic Diseases ◽  
Repetitive Sequences ◽  
Single Copy ◽  
Chromosome 2 ◽  
Insert Size ◽  
Base Pairs ◽  
The Mean

A bank of cloned DNA sequences from the distal half of the short arm of human chromosome 2 was generated by using microdissection and microcloning techniques. DNA was purified from 106 chromosomal fragments, manually dissected from peripheral lymphocytes in metaphase, and cloned into the EcoRI site of lambda gt10. A total of 257 putative recombinants were recovered, of which 41% were found to contain human inserts. The mean insert size was 380 base pairs (median size, 83 base pairs), and fewer than 10% of the clones contained highly repetitive sequences. All single-copy sequences examined were shown to map to the short arm of chromosome 2 by using hybrid panels. This technique provides a rapid method of isolating probes specific to a human subchromosomal region to generate linked markers to genetic diseases for which the chromosomal location is known.

DNA sequence organization in the common Pacific starfish Pisaster ochraceous

1978 ◽  
Vol 56 (11) ◽  
pp. 1048-1054 ◽  
Author(s):  
Michael J. Smith ◽  
Robin Boal
Keyword(s):  
Dna Sequences ◽  
Repetitive Sequences ◽  
Single Copy ◽  
Sea Star ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Reassociation Kinetics ◽  
Short Period ◽  
The Common ◽  
Copy Dna

The sequence arrangement of the genomic DNA from the common sea star Pisaster ochraceous has been examined. Reassociation kinetics at DNA fragment lengths of 300 base pairs (bp) indicate the presence of at least three repetitive components in this DNA. The majority of these repetitive sequences are reiterated over the range from 10 to 100's. Approximately one-third of the nucleotides are found in repetitive sequences. Analysis of the reassociation kinetics of 3000-bp DNA fragments demonstrates the interspersion of repetitive and unique DNA sequences. The hyperchromicity of 3000-bp fragments reassociated to low Cot values (the product of moles of nucleotide per litre and time in seconds), and the size distribution of S1 nuclease resistant DNA duplex in these reassociation products, indicate a short-period interspersion pattern in the starfish genome. Repetitive segments (400 ± 100 bp) are interspersed with longer unique DNA sequences. At a fragment length of 3000 bp the major fraction of the single-copy DNA is found in such an arrangement. In addition to short repetitive segments a substantial portion of the repetitive DNA nucleotides are found in segments excluded by Sepharose CL-2B (≥ 2000 bp). As much as one-quarter of the repetitive sequence nucleotides can be assigned to long segments.

scholarly journals THE DNA OF CAENORHABDITIS ELEGANS

Genetics ◽  
1974 ◽  
Vol 77 (1) ◽  
pp. 95-104
Author(s):  
J E Sulston ◽  
S Brenner
Keyword(s):  
Caenorhabditis Elegans ◽  
Chemical Analysis ◽  
Dna Sequences ◽  
Base Composition ◽  
Nematode Caenorhabditis Elegans ◽  
5S Rna ◽  
Base Pairs ◽  
Small Component ◽  
E Coli ◽  
The Mean

ABSTRACT Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x IO7 base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4s RNA, 110 for 5s RNA, and 55 for (18 + 28)S RNA.

scholarly journals High frequency repeat-induced point mutation (RIP) is not associated with efficient recombination in Neurospora.

Genetics ◽  
1994 ◽  
Vol 138 (4) ◽  
pp. 1093-1103 ◽  
Author(s):  
J T Irelan ◽  
A T Hagemann ◽  
E U Selker
Keyword(s):  
Point Mutation ◽  
Dna Sequences ◽  
High Frequency ◽  
Recombination Frequency ◽  
Single Copy ◽  
Sexual Cycle ◽  
Base Pairs ◽  
Copy Gene ◽  
The Relationship ◽  
Repeat Induced Point Mutation

Abstract Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.

scholarly journals A systematic comparison of eight new plastome sequences from Ipomoea L

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6563
Author(s):  
Jianying Sun ◽  
Xiaofeng Dong ◽  
Qinghe Cao ◽  
Tao Xu ◽  
Mingku Zhu ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Next Generation Sequencing ◽  
Chloroplast Genome ◽  
Dna Sequences ◽  
Repetitive Sequences ◽  
Single Copy ◽  
Next Generation ◽  
Variable Regions ◽  
Chloroplast Genomes ◽  
Generation Sequencing

Background Ipomoea is the largest genus in the family Convolvulaceae. The species in this genus have been widely used in many fields, such as agriculture, nutrition, and medicine. With the development of next-generation sequencing, more than 50 chloroplast genomes of Ipomoea species have been sequenced. However, the repeats and divergence regions in Ipomoea have not been well investigated. In the present study, we sequenced and assembled eight chloroplast genomes from sweet potato’s close wild relatives. By combining these with 32 published chloroplast genomes, we conducted a detailed comparative analysis of a broad range of Ipomoea species. Methods Eight chloroplast genomes were assembled using short DNA sequences generated by next-generation sequencing technology. By combining these chloroplast genomes with 32 other published Ipomoea chloroplast genomes downloaded from GenBank and the Oxford Research Archive, we conducted a comparative analysis of the repeat sequences and divergence regions across the Ipomoea genus. In addition, separate analyses of the Batatas group and Quamoclit group were also performed. Results The eight newly sequenced chloroplast genomes ranged from 161,225 to 161,721 bp in length and displayed the typical circular quadripartite structure, consisting of a pair of inverted repeat (IR) regions (30,798–30,910 bp each) separated by a large single copy (LSC) region (87,575–88,004 bp) and a small single copy (SSC) region (12,018–12,051 bp). The average guanine-cytosine (GC) content was approximately 40.5% in the IR region, 36.1% in the LSC region, 32.2% in the SSC regions, and 37.5% in complete sequence for all the generated plastomes. The eight chloroplast genome sequences from this study included 80 protein-coding genes, four rRNAs (rrn23, rrn16, rrn5, and rrn4.5), and 37 tRNAs. The boundaries of single copy regions and IR regions were highly conserved in the eight chloroplast genomes. In Ipomoea, 57–89 pairs of repetitive sequences and 39–64 simple sequence repeats were found. By conducting a sliding window analysis, we found six relatively high variable regions (ndhA intron, ndhH-ndhF, ndhF-rpl32, rpl32-trnL, rps16-trnQ, and ndhF) in the Ipomoea genus, eight (trnG, rpl32-trnL, ndhA intron, ndhF-rpl32, ndhH-ndhF, ccsA-ndhD, trnG-trnR, and pasA-ycf3) in the Batatas group, and eight (ndhA intron, petN-psbM, rpl32-trnL, trnG-trnR, trnK-rps16, ndhC-trnV, rps16-trnQ, and trnG) in the Quamoclit group. Our maximum-likelihood tree based on whole chloroplast genomes confirmed the phylogenetic topology reported in previous studies. Conclusions The chloroplast genome sequence and structure were highly conserved in the eight newly-sequenced Ipomoea species. Our comparative analysis included a broad range of Ipomoea chloroplast genomes, providing valuable information for Ipomoea species identification and enhancing the understanding of Ipomoea genetic resources.

scholarly journals Chromosomal location of a human alpha interferon gene family.

1982 ◽  
Vol 155 (4) ◽  
pp. 1019-1024 ◽  
Author(s):  
D L Slate ◽  
P D'Eustachio ◽  
D Pravtcheva ◽  
A C Cunningham ◽  
S Nagata ◽  
...  
Keyword(s):  
Human Chromosome ◽  
Gene Family ◽  
Somatic Cell ◽  
Dna Sequences ◽  
Chromosomal Location ◽  
Dna Probe ◽  
Chromosome 9 ◽  
Alpha Interferon ◽  
Somatic Cell Hybrids ◽  
Interferon Gene

To determine the chromosomal location of the human alpha interferon genes, we scored a series of human/rodent somatic cell hybrids for the presence of DNA sequences hybridizing to an alpha 1 interferon DNA probe. The presence of human chromosome 9 in a hybrid correlated with the presence of a family of alpha interferon genes.

scholarly journals The organization and expression of the light gene, a heterochromatic gene of Drosophila melanogaster.

Genetics ◽  
1990 ◽  
Vol 125 (1) ◽  
pp. 129-140 ◽  
Author(s):  
R H Devlin ◽  
B Bingham ◽  
B T Wakimoto
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Molecular Mapping ◽  
Single Copy ◽  
Transcription Unit ◽  
Malpighian Tubules ◽  
Centromeric Heterochromatin ◽  
Chromosome 2 ◽  
Induced Mutations ◽  
Flanking Regions

Abstract The light (lt) gene is located in the centromeric heterochromatin of chromosome 2 of Drosophila melanogaster. This gene is necessary for normal levels of pigmentation in a number of adult and larval tissues and is required for viability. Hybrid dysgenic and X-ray induced mutations have been used to identify the gene and compare its organization to that of euchromatic genes. Molecular mapping of lt mutations and its major transcripts has shown that the lt gene is at least 17 kb. By injecting cosmid clones that include this region into lt mutant embryos, we have defined a 30-kb region that can transiently rescue the pigmentation defect in the Malpighian tubules. The major transcription unit of this gene is comprised of exons that are single copy. It is unusual in its organization in having a heterogeneous array of middle repetitive DNA sequences within its intronic and flanking regions.

scholarly journals 437 DETERMINING GENE COPY NUMBER IN TRANSFECTED CAPRINE FIBROBLAST CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 375
Author(s):  
J. A. Wilson ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Dna Sequences ◽  
Copy Number ◽  
Genomic Dna ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Transgene Copy Number ◽  
Transgenic Organisms ◽  
Plasmid Construct ◽  
The Mean

Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome and by interactions between the transgene and flanking DNA sequences. Very high transgene copy number has also been shown to affect genetic stability in transgenic plants and animals. Two commonly used methods for transfecting cells prior to their use in nuclear transfer (NT) are liposome-mediated transfection and electroporation. Little is known about the transgene copy number or variability of the copy number with these techniques. The objective of this study was to determine transgene copy number after liposome-mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined. Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of a plasmid expressing green fluorescent protein (GFP) under the control of the human elongation factor (hEF) promoter (hEFGFP) used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation with 20 μg of DNA or FuGENE® HD (Roche, Nutley, NJ, USA) reagent with 6 μg of DNA using either a circular or linearized hEFGFP plasmid. Transformed cells were plated at low density in medium containing Geneticin® (Gibco, Grand Island, NY USA). After 10 days of culture, single-cell colonies were isolated and expanded. When cultures reached 1 to 2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1 × 105 cells with Q-PCR. Transgene copy number was calculated from a standard curve of the transgene plasmid. The mean transgene copy number for electroporation circular was 2.7 ± 0.75 (n = 32 colonies) and 1.3 ± 0.65 (n = 19) when using a linear DNA construct. FuGENE HD using a circular plasmid construct generated a mean gene copy number of 0.5 ± 0.11 (n = 14) and 0.64 ± 0.13 (n = 16) for the linear plasmid construct. One-way ANOVA followed by multiple pair-wise comparisons using Tukey’s method showed significant differences when comparing electroporation circular to all other treatments. However, there were no differences when comparing electroporation linear, FuGENE HD circular, and FuGENE HD linear to each other. Because the calculated mean copy number for transfection with FuGENE HD was consistently less than 1, it is assumed that these colonies consisted predominantly of single-copy integrations. Our results indicate that the transfection method can affect gene copy number. Electroporation resulted in multiple but few copies whereas Fugene HD resulted in predominantly single-copy integrations. The probability of transgene mutation with single-copy integration suggests that electroporation is preferable forproducing transgenic animals by NT.

scholarly journals Molecular Markers for the agouti Coat Color Locus of the Mouse

Genetics ◽  
1987 ◽  
Vol 115 (4) ◽  
pp. 747-754
Author(s):  
Michael Lovett ◽  
Zai-yu Cheng ◽  
Estrella M Lamela ◽  
Tohru Yokoi ◽  
Charles J Epstein
Keyword(s):  
Molecular Markers ◽  
Dna Sequences ◽  
Coat Color ◽  
Inbred Mouse ◽  
Inbred Strains ◽  
Single Copy ◽  
Mouse Strains ◽  
Chromosome 2 ◽  
Agouti Locus ◽  
Lethal Yellow

ABSTRACT The agouti (a) coat color locus of the mouse acts within the microenvironment of the hair follicle to control the relative amount and distribution of yellow and black pigment in the coat hairs. Over 18 different mutations with complex dominance relationships have been described at this locus. The lethal yellow (Ay) mutation is the top dominant of this series and is uniquely associated with an endogenous provirus, Emv-15, in three highly inbred strains. However, we report here that it is unlikely that the provirus itself causes the Ay-associated alteration in coat color, since one strain of mice (YBR-Ay/a) lacks the provirus but still retains a yellow coat color. Using single-copy mouse DNA sequences from the regions flanking Emv-15 we have detected three patterns of restriction fragment length polymorphisms (RFLPs) within this region that can be used as molecular markers for different agouti locus alleles: a wild-type agouti (A) pattern, a pattern which generally cosegregates with the nonagouti (a) mutation, and a pattern which is specific to Emv-15. We have used these RFLPs and a panel of 28 recombinant inbred mouse strains to determine the genetic linkage of these sequences with the agouti locus and have found complete concordance between the two (95% confidence limit of 0.00 to 3.79 centimorgans). We have also physically mapped these sequences by in situ hybridization to band H1 of chromosome 2, thus directly confirming previous assignments of the location of the agouti locus.

Sequence organization and developmental expression of an interspersed, repetitive element and associated single-copy DNA sequences in Dictyostelium discoideum

1985 ◽  
Vol 5 (8) ◽  
pp. 2123-2130
Author(s):  
A R Kimmel ◽  
R A Firtel
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Sequence Data ◽  
Single Copy ◽  
Developmental Expression ◽  
Cdna Clones ◽  
Base Pairs ◽  
Sequence Element ◽  
Copy Dna ◽  
Single Copy Dna

We have examined the genomic organization and developmental expression pattern of a short, transcribed, interspersed repeat element and its associated single-copy sequences. We have previously shown that 1% of the polyadenylated [poly(A)+] RNA from vegetative cells contains sequences that hybridize to this repeat. The complementary RNA is heterogeneous in size, and 90% of its mass hybridizes to single-copy DNA. In this study, we examined a series of genomic DNAs and cDNAs derived from poly(A)+ RNAs which are complementary to the repeat. Comparisons of sequence data from various genomic and cDNA clones indicated that (AAC)n X (GTT)n is the common sequence element. The tandem repeat occurred in approximately 100 short segments (approximately 35 to 150 base pairs) per haploid genome interspersed with single-copy DNA. Probes from regions adjacent to this element hybridized to unique restriction fragments on DNA blots and unique poly(A)+ RNA species on RNA blots. The (AAC)n X (GTT)n sequence was asymmetrically transcribed with only (AAC)n sequences represented in RNA. The repeat was localized within the transcribed regions of several genes and 70 base pairs 5' to the transcription initiation site of another gene. Individual (AAC)n-containing RNAs exhibited a developmental pattern of expression suggestive of the coordinate expression of many AAC gene family members.

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