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排序方式: 共有153条查询结果,搜索用时 15 毫秒
1.
David R. Edgell 《Current biology : CB》2009,19(3):R115-R117
2.
A directed nucleotide-sequencing approach for single-stranded vectors based on recloning intermediates of a progressive DNA synthesis reaction 总被引:2,自引:0,他引:2
A simple method for site-directed nucleotide sequencing is presented that uses a novel procedure for generating nested 'deletions' within inserts of single-stranded clones. In this method, single-stranded template, sequencing primer, and the Klenow fragment of Escherichia coli DNA polymerase I are used to initiate progressive DNA synthesis of the entire insert of the clone. By time-dependent sampling and pooling of intermediates from the synthesis reaction a series of nested double-stranded DNA subfragments of the insert can be created. Nested subclones are then produced by S1-endonuclease treatment and oriented subcloning methods. First, smaller quantities of template DNA can be used, equivalent to a fraction of a small DNA sequencing prep. Second, it works with single-stranded M13 phage DNA rather than requiring the preparation of double-stranded replicative form DNA as in ExoIII-based methods. Third, the 'deletions' it generates can span areas of simple nucleotide sequence or secondary structure that often halt digestion in the single-stranded exonuclease-based method. Last, the method is adaptable to a larger variety of insert cloning sites than the ExoIII-based method. The main disadvantage of the method is that, due to the lower efficiency of subcloning larger DNA fragments, subclone inserts larger than 3 kb are generated only infrequently. 相似文献
3.
Conservation throughout mammalia and extensive protein-encoding capacity of the highly repeated DNA long interspersed sequence one 总被引:23,自引:0,他引:23
F H Burton D D Loeb C F Voliva S L Martin M H Edgell C A Hutchison 《Journal of molecular biology》1986,187(2):291-304
We report an investigation of the structure, evolutionary history, and function of the highly repeated DNA family named Long Interspersed Sequence One (L1). Hybridization studies show, first, that L1 is present throughout marsupial and placental mammalian orders. Second, L1 is more homologous within these species than between them, which suggests that it has undergone concerted evolution within each mammalian lineage. Third, on the whole L1 diverges in accordance with the fossil record. This suggests that it arose in each lineage rather by inheritance from a common ancestral family, which was present in the progenitor to mammals, than by cross-species transmission. Alignment of 1.6 X 10(3) bases of primate and mouse L1 DNA sequences shows a predominance of silent mutations within aligned long open reading frames, indicating that at least this part of L1 has produced functional protein. The observation of additional long open reading frames in further unaligned DNA sequences suggests that a minimum of 3.2 X 10(3) bases or at least half of the L1 structure is a protein-coding sequence. Thus L1, which contains about 100,000 members in mouse, is by far the most repetitive family of which a subset comprises functional protein-encoding genes. The ability of the putative protein-encoding regions of mouse L1 to hybridize to L1 homologs throughout the Mammalia implies that these sequences have been subject to conservative selection upon protein function in all mammalian lineages, rather than in a few. L1 is therefore a highly repeated family of genes with both a widespread and an ancient history of function in mammals. 相似文献
4.
An empirical method for the evaluation of the quality of genomic DNA libraries. 总被引:1,自引:0,他引:1 下载免费PDF全文
An empirical method is presented that enables one to determine, with only a relatively small investment of time and materials, whether a lambda genomic DNA library will be a productive source of clones carrying specific sequences. The method provides an indication of the abundance of the sequences in library DNA and indicates whether major DNA rearrangements have occurred during library construction and amplification. 相似文献
5.
6.
Hardies SC; Martin SL; Voliva CF; Hutchison CA d; Edgell MH 《Molecular biology and evolution》1986,3(2):109-125
7.
Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases. 总被引:15,自引:13,他引:2 下载免费PDF全文
D D Loeb C A Hutchison rd M H Edgell W G Farmerie R Swanstrom 《Journal of virology》1989,63(1):111-121
Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites. 相似文献
8.
Cora-Jean S. Edgell Jill E. Haizlip C. Robert Bagnell Joan P. Packenham Paul Harrison Barry Wilbourn Victoria J. Madden 《In vitro cellular & developmental biology. Plant》1990,26(12):1167-1172
Summary Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium
in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the
number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to
senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown
to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific
organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel
Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution
are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor
can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties
are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor.
Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific
organelle in a continuous, vigorously replicating human cell line. 相似文献
9.
A major difference between the divergence patterns within the lines-1 families in mice and voles 总被引:3,自引:0,他引:3
Vanlerberghe F; Bonhomme F; Hutchison CA d; Edgell MH 《Molecular biology and evolution》1993,10(4):719-731
L1 retroposons are represented in mice by subfamilies of interspersed
sequences of varied abundance. Previous analyses have indicated that
subfamilies are generated by duplicative transposition of a small number of
members of the L1 family, the progeny of which then become a major
component of the murine L1 population, and are not due to any active
processes generating homology within preexisting groups of elements in a
particular species. In mice, more than a third of the L1 elements belong to
a clade that became active approximately 5 Mya and whose elements are >
or = 95% identical. We have collected sequence information from 13 L1
elements isolated from two species of voles (Rodentia: Microtinae: Microtus
and Arvicola) and have found that divergence within the vole L1 population
is quite different from that in mice, in that there is no abundant
subfamily of homologous elements. Individual L1 elements from voles are
very divergent from one another and belong to a clade that began a period
of elevated duplicative transposition approximately 13 Mya. Sequence
analyses of portions of these divergent L1 elements (approximately 250 bp
each) gave no evidence for concerted evolution having acted on the vole L1
elements since the split of the two vole lineages approximately 3.5 Mya;
that is, the observed interspecific divergence (6.7%-24.7%) is not larger
than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses
showed no clustering into Arvicola and Microtus clades.
相似文献
10.
DNA sequence organization of the beta-globin complex in the BALB/c mouse 总被引:41,自引:0,他引:41
C L Jahn C A Hutchison S J Phillips S Weaver N L Haigwood C F Voliva M H Edgell 《Cell》1980,21(1):159-168