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1.
cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.  相似文献   

2.
The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.  相似文献   

3.
A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements.  相似文献   

4.
Chick brain actin depolymerizing factor (ADF) is a 19-kDa protein that severs actin filaments and binds actin monomers. We have obtained a cDNA encoding ADF by screening a chick embryo lambda gt11 cDNA library with both a rabbit anti-ADF antiserum and two oligonucleotide probes. Several non-full-length clones of 636 bases and one full-length clone of 1886 bases were isolated and sequenced. The full-length cDNA encodes a protein of 165 amino acids with a calculated molecular weight of 18,520. The deduced amino acid sequence shows 73% identity with the porcine brain actin binding protein cofilin. The coding region of the ADF cDNA has been placed in an expression vector, and the resulting protein shows immunoreactivity with an anti-ADF antiserum but not with an anti-cofilin antibody. The expressed ADF has been purified and has an actin depolymerizing activity identical with that of brain ADF. Like cofilin, ADF contains a sequence similar to the nuclear transport signal sequence of the SV40 large T antigen and a calcium/calmodulin-dependent protein kinase II phosphorylation consensus sequence. Northern blots of both embryonic chick brain and muscle RNA revealed two ADF mRNAs of length 2.1 and 0.9 kilobases. Southern blots suggest that the ADF gene is present in a single copy within the chicken genome. ADF contains regions of homology with other actin binding proteins including tropomyosin, gelsolin, and depactin.  相似文献   

5.
A simple method is described for the construction of subtracted cDNA libraries. The technique was used to create a human pancreatic tumor cDNA library that was screened using either hybridization with cDNA probes or antibodies. cDNA from a well-differentiated tumor cell line (CD-11) was subtracted against RNA from an undifferentiated tumor cell line (Panc-1). The subtracted cDNA was purified from RNA-cDNA hybrids by oligo-dA cellulose affinity chromatography. Single-stranded subtracted cDNA was used as a template for random primed second-strand synthesis using the Klenow's fragment of DNA polymerase. After ligation with Eco R1 adapters, cDNA was inserted into lambda gt11. A library of 140,000 primary pfu was obtained that contained 92% recombinants. A small portion of this library (40,000 pfu) was subjected to probe screening with a mucin cDNA probe known to be differentially expressed by CD-11 cells. The ratio of mucin cDNA clones to actin cDNA clones was increased by greater than 300-fold in the subtracted cDNA library compared to a standard cDNA library from the same cell line. The absolute number of mucin cDNA clones per 40,000 pfu was also increased 32-fold in the subtracted library. Pancreatic tumor mucin cDNAs were also identified in the subtracted library by antibody screening. The subtraction procedure yielded a 50-fold enrichment in differentially expressed cDNA detected by antibodies, compared to a nonsubtracted library from the same cell line.  相似文献   

6.
cDNA clones encoding the actin filament severing protein severin from Dictyostelium discoideum were isolated from a cDNA library in lambda gt 11 using monoclonal antibodies. Comparison of the deduced amino acid sequence with the sequence of a severin peptide indicated that the complete coding region of severin is contained in the isolated clones. Severin, a 39.9-kDa protein, is encoded by one gene in D. discoideum. An mRNA of approximately 1.4 kilobases is present throughout the developmental cycle of D. discoideum. The amino acid sequence of severin contains a region highly homologous to a conserved sequence in villin and gelsolin, two proteins of similar function isolated from vertebrates. This homologous region is believed to participate in the actin filament severing activity of these proteins. Comparison of the severin sequence to the entire gelsolin sequence shows remarkable homologies pointing to a common origin from an ancestral gene from which gelsolin has been derived by a duplication.  相似文献   

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Monoclonal antibodies were raised against a protein with a molecular mass of 24 kDa that has been described as a membrane-associated, actin binding protein from Dictyostelium discoideum [( 1985) J. Cell Biol. 100, 727-735]. Using these monoclonal antibodies we isolated from a lambda gt11 expression library cDNA clones coding for this protein. The cDNA deduced amino acid sequence revealed the presence of an unusual carboxy-terminus which has homologies to the C-termini of Octopus rhodopsin and synaptophysin. This part of the protein sequence contains 5 direct repeats with the motif GYP (P)Q(P). Southern and Northern blots showed that this sequence is present in a series of Dictyostelium genes transcribed in all stages of development.  相似文献   

10.
Studies of gene expression during blastocyst formation in mouse preimplantation development have been limited by the amount of RNA available per embryo. Our present approach to this problem has been to construct a large, representative, blastocyst cDNA library in lambda gt11. Random hexadeoxynucleotides were used as primers with total blastocyst RNA serving as template. RNA collected from 4,100 32-64 cell embryos was used to generate a library with an initial size of 30 X 10(6) recombinants. By using clone frequency as a measure of relative mRNA abundance, our data support previous work on the relative and absolute amounts of actin, histone H2a, and intracisternal A particle. Furthermore, we provide estimates for the abundance of cytokeratin endo A, cytokeratin endo B, and beta-tubulin from clone frequency data. Insert sizes for isolated clones range from 200 bp to 3.6 kb with full-length or near-full-length insert sizes for selected clones, indicating that random primer methods generate cDNAs which can represent a significant portion of the mRNA. We have so far characterized products whose abundance is equal to or greater than 0.002% of total RNA. This library offers the potential for the analyses of presumptive regulatory gene products in the mouse preimplantation embryo which are represented as low abundance (less than 1% of mRNA) RNAs.  相似文献   

11.
用改进的异硫氰酸胍-苯酚-氯仿抽提法提取了豌豆卷须总RNA并纯化了mRNA,合成双链cDNA后加上人工接头,Sepharose2B柱层析去掉小片段,最后连入载体λ-ZAPⅡ并用噬菌体包装蛋白进行体外包装,经稀释测定出含有重组子的文库大小为9.2×10~5.原位杂交筛选出了肌动蛋白阳性克隆。  相似文献   

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Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.  相似文献   

14.
A cDNA library of ovine pituitary DNA in plasmid pBR322 has been constructed by conventional methods with certain modifications. The library was screened using partial cDNAs for ratα-subunit and LHβ. We have isolated cDNA clones for ovineα-subunit and LHβ. The identification of these clones was confirmed by partial sequencing. The clones bear about 80% sequence homology with the respective rat cDNAs in the sequenced regions and hybridize with the rat clones in 5 X SSC at 55°C. The ovine LHβ clone has an insert of about 650 bp and selects an RNA of about 750 bases in a northern blot. The α-subunit cDNA clone has an insert of about 550 bp; it has two internalPst I sites and thus shows restriction-based differences from ratα-subunit cDNA, which does not have anyPst I site.  相似文献   

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16.
A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.  相似文献   

17.
A cDNA library of Plasmodium falciparum (Colombian strain FCB2) asexual stage was constructed in the lambda ZipLox vector. The lambda ZipLox library and a lambda ZAPII (Dd2 strain) were screened for genes coding for proteins that bind with or are related to calmodulin (CaM). Screening was accomplished with Hot start PCR assays and hybridization with radiolabeled probes. Actin I, CaM, glutamate synthase (GOGAT) and the three myosin clones--Pfmyo A, Pfmyo B and Pfmyo C--were identified. The clones coding for actin I, CaM and GOGAT were retrieved from the lambda ZipLox library, and the GOGAT and Pfmyo A clones from the lambda ZAP II library. The GOGAT clone contained an insert of 2,413 base pairs corresponding to 24.8% of the reported sequence. The Pfmyo A insert was 2,457 base pairs long, and represented the complete mRNA coding for this gene. Finally, the first report of a complete cDNA clone containing the P. falciparum myosin A is presented.  相似文献   

18.
Four cDNA clones coding for different Artemia actin isoforms have been isolated. Three of the clones contain the complete coding sequences while the fourth one lacks 145 bases, coding for the 49 amino terminal amino acids of the protein. The amino acid sequences predicted for the four actin isoforms identified are highly homologous to insect actins as well as to vertebrate cytoplasmic actins. The four identified cDNA clones code for mRNAs of 5.2, 1.9, 1.6 and 1.8 kb, respectively, whose expression is regulated during development. Three of the actin mRNAs are present in cryptobiotic embryos while the other is not. The steady-state levels of all four mRNAs increase during development to reach maximal levels by 10-15 hours of development and decrease thereafter. The total number of actin genes encoded in the Artemia genome has been estimated as 8 to 10 by Southern analysis of total DNA.  相似文献   

19.
Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA. A library of recombinant clones was constructed by inserting the dsDNA into the Pst1 site of pBR322 plasmid and transformation of E. coli chi 1776 host. Clones bearing sequences coding for tubulin and actin were identified and characterized.  相似文献   

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