用抑制差减杂交法分离和克隆梭梭幼苗受渗透胁迫诱导相关基因的cDNA片段

以Hoagland溶液培养的梭梭幼苗(H)为对照群体,甘露醇处理的梭梭幼苗(M)为目标群体,进行抑制差减杂交.用经过H cDNA差减的M cDNA构建了一个含有大约400个独立克隆的差减文库;采用差减前的H cDNA和M cDNA以及正向/反向差减杂交后的cDNA为模板标记探针,对随机挑取的100个重组质粒进行差示筛选,获得了21个阳性候选克隆.从这些阳性候选克隆中随机挑取了8个进行Northern blot分析,证实其中3个候选克隆代表了在M中特异表达或表达增强的基因,序列分析和同源性比较表明它们与逆境胁迫有关;而另外5个候选克隆无Northem杂交信号,推测它们为低丰度转录本.     

Two arylamine N-acetyltransferases from chicken pineal gland as identified by cDNA cloning

A cDNA library prepared from the poly(A)-rich RNA of the chicken pineal gland obtained at night was screened with the 32P-labeled cDNA of arylamine N-acetyltransferase from the chicken liver recently isolated in this laboratory. Two positive clones (p-NAT-3 and p-NAT-10) that cross-hybridized with the liver cDNA were isolated. The cDNAs did not cross-hybridize each other under a high stringency, indicating that they corresponded to different mRNAs. When the cDNAs were inserted into an expression vector pcDL1 under the control of the early promoter of simian virus 40 and introduced into Chinese hamster ovary cells, both cDNAs expressed arylamine N-acetyltransferase activity in the transfected cells. The nucleotide sequences of the cDNAs were determined, from which amino acid sequences were deduced. Both cDNAs coded for 290 amino acids. Similarities in amino acid sequences were about 60% between p-NAT-3, p-NAT-10 and liver N-acetyltransferases. Poly(A)-rich RNA blot hybridization analysis indicated that p-NAT-3 cDNA detected a 2.2-kb band with the poly(A)-rich RNAs from the brain, gut and, less intensively, spleen, liver and kidney, while p-NAT-10 cDNA hybridized only with the poly(A)-rich RNA from the kidney. Neither cDNA detected any hybridization band with the poly(A)-rich RNA from the pineal gland, suggesting that the contents were low. Genomic Southern blot hybridization analysis showed that p-NAT-3, p-NAT-10 and liver N-acetyltransferases were encoded in a separate single gene. The properties of the enzymes expressed in the transfected cells were compared with N-acetyltransferases from the pineal gland, brain and kidney. On a DEAE-cellulose column, the kidney and p-NAT-10 enzymes appeared in the effluent fraction, whereas the brain and p-NAT-3 enzymes were eluted from the column with a gradient elution at 0.08 M NaCl. The supernatant of the pineal gland obtained in the daytime showed two peaks appearing in the effluent fraction and the eluate fraction at 0.08 M NaCl. The substrate specificity of these enzymes were examined with p-phenetidine, 2-aminofluorene, tryptamine and phenylethylamine as substrates. All the enzymes preferred arylamines to arylalkylamines, indicating that both p-NAT-3 and p-NAT-10 cDNAs encoded arylamine N-acetyltransferases.     

A simple, effective method for the construction of subtracted cDNA libraries

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.     

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.     

A nested set of eight RNAs is formed in macrophages infected with lactate dehydrogenase-elevating virus.

Total RNA was extracted from primary cultures of mouse macrophages isolated from 10-day-old mice 6 to 12 h postinfection with lactate dehydrogenase-elevating virus (LDV). Poly(A)+ RNA was extracted from spleens of 18-h LDV-infected mice. The RNAs were analyzed by Northern (RNA) blot hybridization with a number of LDV-specific cDNAs as probes. A cDNA representing the nucleocapsid protein (VP-1) gene located at the 3' terminus of the viral genome (E. K. Godeny, D. W. Speicher, and M. A. Brinton, Virology 177:768-771, 1990) hybridized to viral genomic RNA of about 13 kb plus seven subgenomic RNAs ranging in size from about 1 to about 3.6 kb. Two other cDNA clones hybridized only to the four or five largest subgenomic RNAs, respectively. In contrast, two cDNAs encoding continuous open reading frames with replicase and zinc finger motifs hybridized only to the genomic RNA. The replicase motif exhibited 75% amino acid identity to that of the 1b protein of equine arteritis virus (EAV) and 44% amino acid identity to those of the 1b proteins of coronaviruses and Berne virus. Combined, the results indicate that LDV replication involves formation of a 3'-coterminal-nested set of mRNAs as observed for coronaviruses and toroviruses as well as for EAV, with which LDV shares many other properties. Overall, LDV, like EAV, possesses a genome organization resembling that of the coronaviruses and toroviruses. However, EAV and LDV differ from the latter in the size of their genomes, virion size and structure, nature of the structural proteins, and symmetry of the nucleocapsids.     

Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA

A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.     

Cytoplasmic RNA extraction from fresh and frozen mammalian tissues

The quality of collections of expressed sequence tags andfull-length cDNAs is adversely affected by the presence of "junk" clones derivedfrom unspliced or partially spliced RNAs present in conventional total RNA preparations. One can overcome this problem by using intact cytoplasmic RNA to create cDNA libraries, but the methods in the literature that describe the preparation of RNA only work well for extracting cultured cells. Cell lines are not as diverse as one would like, and to clone comprehensive sets of human and model organism full-length cDNAs, libraries have to be prepared from tissue samples. Thus, we have developed a robust and inexpensive method that allows intact cytoplasmic RNA to be extracted from both fresh and frozen mammalian tissues. A mouse full-length, cap-trapped cDNA library prepared with RNA using this new procedure had excellent characteristics.     

Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families

A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stage-specific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.     

Albumin is encoded by 2 messenger RNAs in Xenopus laevis

A cDNA clone library was prepared from liver poly(A) RNA pf non-estrogenized Xenopus laevis. Albumin coding sequences were screened by hybridization to a cDNA prepared from poly(A) RNA enriched by sucrose density gradient centrifugation, and by a sensitive solid-phase radioimmunoassay to detect clones that contain templates for albumin antigenic determinants. Nine clones were obtained by this approach, and all but one have the cDNA inserted in phase with the beta-lactamase gene of pBR322. Mapping of these clones with restriction endonucleases yielded 2 distinct patterns, suggestive of heterogeneity in the coding sequences. This was confirmed by heteroduplex analyses of hybrids formed between clones representative of each of the 2 classes. Both classes of albumin cDNA clones were used to select mRNAs of the same size (2.3kb) that code for peptides that are indistinguishable by SDS gel electrophoresis. Examination of the organization of the albumin genes by blot hybridization of the cDNA clones to restriction fragments of Xenopus DNA failed to detect any differences at the genomic level. The considerable diversity of the albumin cDNAs is suggestive of a multiplicity of albumin genes, rather than differential processing of a common precursor RNA.     

Analysis of male sterile mutations in the mouse using haploid stage expressed cDNA probes

A differential hybridization screening procedure has identified cDNAs which correspond to RNAs which are expressed in mouse testis and at lower levels in liver and spleen. The sensitivity of this procedure is such that approximately 0.5% of 1.4 X 10(4) cDNA clones are revealed as "testis specific". We have focused on ten cDNA clones which have been used to identify RNAs expressed in the haploid phase of spermatogenesis. Using Northern blots to analyse RNA isolated from the testes of mutant mice (Tfm/Y and Sxr/+) blocked at specific stages in spermatogenesis or RNA from sexually immature mice, 8 clones have been identified which correspond to RNAs expressed uniquely or at much higher levels in meiotic or post meiotic cells.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.     

cDNA selection with YACs

Identification of expressed sequence tags (ESTs) in large genomic segments is an important step in positional cloning and genomic mapping studies. A simple and efficient polymerase chain reaction (PCR)-based approach is described here to identify coding sequences in large genomic fragments of DNA cloned in vectors such as yeast artificial chromosome (YAC) vectors. The method is based on blocking of sequences such as repetitive and GC rich sequences in the genomic DNA immobilized on nylon paper discs prior to hybridization of the discs to cDNA library, and recovery of the selected cDNAs by the PCR. Single or multiple cDNA libraries can be used in the selection procedure. The procedure has been used successfully also with total yeast DNA containing a YAC.     

Purification of cDNA complementary to sea urchin histone mRNA.

Complementary DNA (cDNA) was transcribed from a polyadenylated sea urchin histone mRNA preparation isolated by density gradient centrifugation. By hybridization, this cDNA was shown to be extensively contaminated (85% of hybridizable cDNA) with DNA complementary to RNA derived from the large ribosomal subunit. Purification of a mRNA specific cDNA fraction was achieved by hybridization of purified rRNA to cDNA followed by fractionation on hydroxyapatite. After further purification to remove nonhybridizable cDNA our purified cDNA showed only 8% hybirdization to rRNA.     

Synthesis of complementary DNA from lens mRNA with RNA-dependent DNA polymerase

Lens 10S and 14S mRNAs, isolated by zonal centrifugation, were shown to function as templates for the synthesis of complementary DNA (cDNA) with RNA-dependent DNA polymerase of avian myeloblastosis virus (AMV). The cDNA products, synthesized with the lens 10S and 14S mRNA templates, gave sedimentation constants of 7.6S and 8.3S, respectively. The complementarity of the cDNAs to their specific RNA templates was demonstrated by hybridization experiments.