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1.
Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments
containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts
were analyzed by Southern blot analysis using the following32P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)16 simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as
a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated
the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone
genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined.
These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions
despite marked differences in their overall structure and pattern of expression. 相似文献
2.
Carol M.. Hamilton Anne Frary Yimin Xu Steven D.. Tanksley Hong-Bin Zhang 《The Plant journal : for cell and molecular biology》1999,18(2):223-229
This is the first report of large insert genomic DNA libraries constructed in a binary-BAC (BIBAC) vector. Genomic DNA libraries containing approximately 4.6 haploid nuclear genomic equivalents were constructed for Lycopersicon esculentum (cv. Mogeor) and Lycopersicon pennellii (LA716) in the BIBAC2 vector. The L. esculentum library has an average insert size of 125 kb and is comprised of 42 272 individual colonies stored as frozen cultures in a 384-well format (108 plates). The L. pennellii library has an average insert size of 90 kb and is comprised of 53 760 individual clones (140 384-well plates). In each of the libraries, it is estimated that 90% of the colonies contain genomic DNA inserts. The composition of the L. esculentum and L. pennellii libraries was determined by analyzing a series of randomly selected clones. The L. esculentum library was surveyed for clones containing chloroplast DNA (1.4%), mitochondrial DNA (0.012%) and repetitive DNA motifs. BIBAC clones that may contain a gene of interest can be identified from these libraries by colony hybridization with homologous or heterologous probes or by PCR pooling techniques. Once identified, BIBAC genomic DNA library clones are immediately suitable for Agrobacterium tumefaciens-mediated plant transformation. 相似文献
3.
Kazuhiro Yoshikawa Masao Seto Ryuzo Ueda Yuichi Obata Kunihiro Notake Takashi Yokochi Toshitada Takahashi 《Immunogenetics》1991,33(5-6):352-360
The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites — 122 base pairs (bp) to — 38 bp from ATG translation initiation site were demonstrated. The promoter region had high G+C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs. 相似文献
4.
Fiona M. Mellon Peter F. R. Little Lorna A. Casselton 《Molecular & general genetics : MGG》1987,210(2):352-357
Summary The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu+ transformants was not possible because of the toxicity of the acetate selection medium. Acu+ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp+ transformants were cotransformed to Acu+. A physical analysis of 40 Acu+ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses. 相似文献
5.
DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.by R. Appels 相似文献
6.
Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. 总被引:342,自引:0,他引:342
Treatment of Ltk?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk+ clones with a frequency of 10?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk+ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation. 相似文献
7.
Construction,characterization, and screening of a transformation-competent artificial chromosome library of peach 总被引:7,自引:1,他引:6
Feng Shan Liang Kai Chun Zhang Zhan Wang Yu Ji Liang Yang Xiao Ming Zhang Jin De Min Bin Wang 《Plant Molecular Biology Reporter》2004,22(1):37-48
As a genome model of fruit trees, peach (Prunus persica [L.] Batch) has advantages for studying structural and functional genomics. Okubo, a traditional peach variety used as a
parent in Asian peach breeding, displays economically valuable agronomic traits. To develop an efficient platform for peach
gene cloning and genomic research, a large-insert genomic DNA library of Okubo was constructed in a transformation-competent
artificial chromosome (TAC) vector, pYLTAC7, which can accept and stably maintain large genomic DNA fragments in bothEscherichia coli andAgrobacterium tumefaciens. The TAC library contains 41,472 recombinant clones with an average insert size of approximately 42 kb, and it is equivalent
to 6 haploid peach genomes. The TAC library was stored in 2 ways: one copy as frozen cultures in 108 pieces of 384-well plates
and another copy as bulked pools in 36 pieces of 96-well plates, each well containing 12 individual clones. The lack of hybridization
signal to chloroplast and mitochondrial genes indicated that the TAC library had no significant cytoplast organelle DNA contamination.
TAC clones were stable inE. coli cells until generation 100 and stable in bothE. coli andA. tumefaciens. Twenty-one clones containing the polygalacturonase-inhibiting protein (PGIP) gene were detected by using pooled PCR in the
TAC library. Positive clones can be used for peach PGIP gene cloning and functional analysis. The library is well suited for
gene cloning and genetic engineering in peach. 相似文献
8.
9.
10.
Background
Retrotransposons have been extensively studied in plants and animals and have been shown to have an impact on human genome dynamics and evolution. Their ability to move within genomes gives retrotransposons to affect genome instability.Methods
we examined the polymorphic inserted AluYa5, evolutionary young Alu, in the progesterone receptor gene to determine the effects of Alu insertion on molecular environment. We used mono-allelic inserted cell lines which carry both Alu-present and Alu-absent alleles. To determine the epigenetic change and gene expression, we performed restriction enzyme digestion, Pyrosequencing, and Chromatin Immunoprecipitation.Results
We observed that the polymorphic insertion of evolutionally young Alu causes increasing levels of DNA methylation in the surrounding genomic area and generates inactive histone tail modifications. Consequently the Alu insertion deleteriously inactivates the neighboring gene expression.Conclusion
The mono-allelic Alu insertion cell line clearly showed that polymorphic inserted repetitive elements cause the inactivation of neighboring gene expression, bringing aberrant epigenetic changes. 相似文献11.
The CD4 and CD8 molecules are rapidly phosphorylated following exposure of CD4+ or CD8+ human cytotoxic T lymphocytes (CTL) clones to B-lymphoblastoid cell lines bearing the relevant target alloantigens. Treatment
of CD4+ or CD8+ CTL clones with phorbol myristate acetate (PMA), phytohemagglutinin, or mitogenic combinations of CD2-specific antibodies
also resulted in CD4 or CD8 phosphorylation. Down-regulation of the surface expression of these molecules could be demonstrated
in both CD4+ and CD8+ clones following exposure to the relevant alloantigen or PMA. Parallel experiments were conducted using mouse L cells in
which the human CD4 or CD8 antigens were stably expressed. Exposure of these transfectants to PMA induced rapid phosphorylation
of the CD4 and CD8 molecules. As in CD4+ CTL clones, rapid modulation of the CD4 antigen could be demonstrated in L cells following PMA treatment. In contrast, there
was no demonstrable down-regulation of the CD8 antigen in PMA-treated CD8+ L cell transfectants. These studies demonstrate a significant differential property of the CD4 and CD8 antigens and suggest
that down-regulation of the CD8 antigen may require its expression in a T-cell environment and/or the association of CD8 with
the T-cell receptor or other T cell-specific molecules. 相似文献
12.
Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This
gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H+-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
We generated specific rabbit polyclonal antibody against the Mr=100,000 H+-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive
protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot
cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene
for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential
for use of recombinant DNA technology in plant mineral transport research are discussed. 相似文献
13.
Maize DNA enrichment by representational difference analysis in a maize chromosome addition line of oat 总被引:1,自引:0,他引:1
Z. J. Chen Ronald L. Phillips Howard W. Rines 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(3):337-344
The recent recovery of maize (Zea mays L.) single-chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses has provided novel source materials for the potential isolation of maize chromosome-specific
sequences for use in genetic mapping and gene cloning. We report here the application of a technique, known as representational
difference analysis (RDA), to selectively isolate maize sequences from a maize chromosome-3 addition line of oat. DNA fragments
from the addition line and from the oat parent were prepared using BamHI digestion and primer ligation followed by PCR amplification. A subtractive hybridization technique using an excess of the
oat parental DNA was employed to reduce the availability for amplification of DNA fragments from the addition lines that were
in common with the ones from the oat parental line. After three rounds of hybridization and amplification, the resulting DNA
fragments were cloned into a plasmid vector. A DNA library containing 400 clones was constructed by this method. In a test
of 18 clones selected at random from this library, four (22%) detected maize-specific repetitive DNA sequences and nine (50%)
showed strong hybridization to genomic DNA of maize but weak hybridization to genomic DNA of oat. Among these latter nine
clones, three detected low-copy DNA sequences and two of them detected DNA sequences specific to chromosome 3 of maize, the
chromosome retained in the source maize addition line of oat. The other eight out of the 13 clones that had strong hybridization
to maize DNA detected repetitive DNA sequences or high-copy number sequences present on most or all maize chromosomes. We
estimate that the maize DNA sequences were enriched from about 1.8% to over 72% of the total DNA by this procedure. Most of
the isolated DNA fragments detected multiple or repeated DNA sequences in maize, indicating that the major part of the maize
genome consists of repetitive DNA sequences that do not cross-hybridize to oat genomic sequences.
Received: 18 November 1997 / Accepted: 12 March 1998 相似文献
14.
We have cloned cDNA copies of in vitro adenylated 7S RNA of HeLa cells. The most representative clones in the library contain DNA fragments copied from the 7SL and 7SK small RNAs. The two classes of recombinants share no homology. The 7SL RNA contains at the 5' end of the molecule sequences homologous to the Alu sequence family. Hybridization to human genomic DNA shows that the 7SL and 7SK clones are homologous to two different families of repetitive sequences. 相似文献
15.
A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische
Forschung (IGF) BAC library, consists of 10 752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100 kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA
and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid
DNA, 3.2% clones with the 180 bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA
repeat. With its extensive genome coverage, its rather uniformly sized inserts (80 kb <85% <120 kb) and low contamination
with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.
Received: 26 November 1997 / Accepted: 19 February 1998 相似文献
16.
Shibata F Matsusaki Y Hizume M 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2005,110(7):1253-1258
Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8–76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats. 相似文献
17.
Mauro S. Sandrin K. Erin Lovering George Tachas Peter R. Collins Ian F. C. McKenzie 《Immunogenetics》1987,25(5):279-283
Human DNA was transfected into mouse L cells and tk+ HuLy-m2+ (= CD7+) transfectants isolated after growth in hypoxanthine, aminopterin, thymidine medium and repeated cloning. After several cycles of transfection, > 90% of HuLy-m2+ L cells could be detected, by rosetting and by cytofluorography, which showed the transfectants to have a density of CD7 two to five times that found on peripheral blood lymphocytes. Despite this, the 37 kd CD7+ dimer could only be identified with difficulty using cell-surface radioiodination and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques. An antiserum was produced (C3H anti-HuLy-m2+ L cells) which, after absorption, was shown to react with HuLy-m2 antigens present on human thymocytes and lymphocytes and on CD7+ transfected L cells.Abbreviations BSA
bovine serum albumin
- DME
Dulbecco's modified Eagle's medium
- EDTA
ethylenediamine-tetraacetate
- HAT
hypoxanthine, aminopterin, thymidine
- HSV
herpes simplex virus
- PBL
peripheral blood lymphocyte
- PBS
phosphate-buffered saline
- RFC
rosette-forming cell
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- tk
thymidine kinase 相似文献
18.
A. V. Sekridova A. M. Varizhuk O. N. Tatarinova V. V. Severov N. A. Barinov I. P. Smirnov V. N. Lazarev D. V. Klinov G. E. Pozmogova 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2017,11(1):62-71
We report results of the first systematic study of conformational polymorphism of G-rich DNA fragments of Alu-repeats. Alu retrotransposons are primate-specific short interspersed elements. Using the Alu sequence of the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we have determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites—PQSAlu), and demonstrated by bioinformatics methods that these sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 bp). These sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element of the human bcl2 gene (304 bp) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and their absence in the case of Alu-PQS have been shown using DMS-footprinting and atomic force microscopy (AFM). Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions of the reporter gene have been prepared, and their regulatory effects have been compared during transfection of НЕК293 and HeLa cells. We suggest that the dynamic study of the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements responsible for the development of many oncological and neurodegenerative diseases. 相似文献
19.
The human cyclophilin gene was isolated from a genomic library derived from leucocyte DNA and sequenced. The gene contains five exons and four introns. The amino acid sequence deduced from the exons matches perfectly the one previously determined from the T-cell cyclophilin cDNA. A TATA box is visible in the promoter region and putative Sp1 binding sites are also found there as well as in the first intron. Six members of the middle repetitive Alu gene family are present in one or other orientation in the non-coding regions of the cyclophilin gene. Hybridisation of genomic DNA to probes derived from the promoter region or the first intron indicates that the cyclophilin gene is present as a single copy in the human haploid genome. Seven other cyclophilin-related DNA clones isolated from the same library were also characterized. They show a high degree of similarity to the cyclophilin cDNA and are colinear to it. However, multiple genetic lesions, often including deletion and/or insertion events which modify the reading frame, are found in these clones which are therefore likely to represent processed pseudogenes. 相似文献
20.
Species-specific EcoRI repetitive elements of at least 16 kb in length are present in Lupinus luteus
T. Sakowicz 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1992,85(2-3):303-308
Summary A genomic DNA library of Lupinus luteus cv. Ventus was constructed in the phage vector EMBL3 using Mb oI-digested DNA. Screening with a 1070 bp labelled repetitive unit from L. luteus yielded several DNA clones. The repetitive family is composed of elements whose length is at least 16 kb. The average copy number of the cloned fragments is 5.0 × 104 per haploid genome and constitutes approximately 3% of the total L. luteus genome. The homologous repeats were found in all ten cultivars of L. luteus tested but were not detected in two cultivars each of the closely related species Lupinus albus and Lupinus angustifolius. The EcoRI family fragments could thus be considered as species-specific DNA elements. These fragments may be useful as molecular markers in the genetic manipulation of L. luteus. 相似文献