Identification of bacterial clones that encode cow's caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant DNA plasmids carrying cDNA fragments of bovine caseins in the cDNA library from rRNA of bovine mammary glands. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter paper. Antigens covalently bound to the CNBr-activated paper or bound to the nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. Four clones were positive among 5400 bacterial clones of the cDNA library--al, b2, b5, h7. Molecular weights of chimeric proteins pre-beta-lactamase:casein synthesized in Escherichia coli were determined by immunoblotting. Colony hybridization and DNA sequence analysis showed that clone b5 contained cDNA fragment of bovine kappa-caseins and clone h7 cDNA fragment of beta-casein. The last clone was designated pKcas beta-7.     

人ZP3基因的RT-PCR cDNA克隆

目的：研究人ZP3基因的结构及构建人ZP3基因原核表达系统。方法：从人卵巢组织中分离出mRNA并以此作为模版,通过RT—PCR扩增出人ZP3基因cDNA片段,然后将其克隆在pUC18质粒上,并对克隆片段进行序列分析。结果：共克隆到ZP3-A（1300bp）、ZP3-B（1180bp）、ZP3-C（1200bp）和ZP3-D（1080bp）4种不同长度的人ZP3基因cDNA片段,对其中最长的ZP3-A片段的测序结果表明,它包含了人ZP3基因阅读框内的全部序列,与NCBI Sequence Viewer中公布的人ZP3 mRNA序列（NM-007155）相比较,在1275bp长的编码区内只有一个碱基不同,两者同源性达到99．92％。结论：本研究克隆到的ZP3-A cDNA片段确是人ZP3基因无疑。     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-α) and fibroblast (HuIFN-β) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-α clone, 201, has a nucleotide sequence different from published HuIFN-α sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-α in Escherichia coli.     

Establishment of Novel Monoclonal Fabs Specific for Epstein-Barr Virus Encoded Latent Membrane Protein 1

<正>Dear Editor,Epstein-Barr virus (EBV, also termed human herpesvirus-4) was the first identified human tumor virus. Since its discovery in 1964, studies have shown that EBV infects over 90%of all people by the time they are adults(Williams and Crawford 2006). EBV infection can result in     

Carcinoembryonic antigen gene family members in submandibular salivary gland: demonstration of pregnancy-specific glycoproteins by cDNA cloning

We have recently shown that human submandibular salivary gland and saliva contain a number of glycoproteins belonging to the carcinoembryonic antigen (CEA) gene family. The members of the CEA family can be divided into the CEA subgroup and the pregnancy specific beta 1 glycoprotein (PSG) subgroup. The latter glycoproteins are abundant in placenta and fetal liver. Here we report that PSG's are expressed in normal adult submandibular salivary gland. Thus, cDNA cloning and sequencing gave two clones (SG5 and SG9) which coded for glycoproteins with a domain arrangement of N-A1-A2-B2-C and a third clone (SG8) which coded for a glycoprotein with a domain arrangement of N-A1-B2-C. SG5 is identical to PSG3, and SG9 to PSG1d, while SG8 most probably corresponds to PSG2. The 3' untranslated regions of the different members of the PSG subgroup contain highly homologous segments, suggesting a common evolutionary origin.     

The 5′ untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3′ end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5′ untranslated region (5′ UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUKl is identical to the 5′ UTR of ACaM-2 and that the 5′ UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0 ×10⁴ clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indicajaponica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

Human apolipoprotein A-II: complete nucleic acid sequence of preproapoA-II

The complete cDNA nucleic acid sequence of preproapolipoprotein (apo) A-II, a major protein constituent of high density lipoproteins, has been determined on clones from a human liver ds-cDNA library. Clones containing ds-cDNA for apoA-II were identified in the human liver ds-cDNA library using synthetic oligonucleotides as probes. Of 3200 clones screened, 4 reacted with the oligonucleotide probes. The DNA sequence coding for amino acids ?17 to +17 of apoA-II were determined by Maxam-Gilbert sequence analysis of restriction fragments isolated from one of these clones, pMDB2049. The remainder of the cDNA sequence was established by sequence analysis of a primer extension product synthesized utilizing a restriction fragment near the 5'-end of clone pMDB2049 as primer with total liver mRNA. The apoA-II mRNA encodes for a 100 amino acid protein, preproapoA-II that has an 18 amino acid prepeptide and a 5 amino acid propeptide terminating with a basic dipeptide (Arg-Arg) at the cleavage site to mature apoA-II.     

Structure of the rabbit tc-casein encoding gene: expression of the cloned gene in the mammary gland of transgenic mice

The rabbit κ-casein (κ-Cas) encoding gene has been isolated as a series of overlapping DNA fragments cloned from a rabbit genomic library constructed in bacteriophage λEMBL3. The clones harboured the 7.5-kb gene flanked by about 2.1 kb upstream and 9 kb downstream sequences. The cloned gene is the most frequently occurring of two κ-Cas alleles identified in New Zealand rabbits. Comparison of the corresponding domains in rabbit and bovine κ-Cas shows that both genes comprise 5 exons and that the exon/intron boundary positions are conserved whereas the introns have diverged considerably. The first three introns are shorter in the rabbit, the second intron showing the greatest difference between the two species: 1.35 kb instead of 5.8 kb in the bovine gene. Repetitive sequence motives reminiscent of the rabbit C type repeat and the complementary inverted C type repeat were identified in the fourth and first introns, respectively. Transgenic mice were produced by microinjecting into mouse oocytes an isolated genomic DNA fragment which contained the entire κ-Cas coding region, together with 2.1-kb 5′ and 4.0-kb 3′ flanking region. Expression of transgene rabbit κ-Cas mRNA could be detected in the mammary gland of lactating transgenic mice and the production of rabbit κ-Cas was detected in milk using species-specific antibodies. The cloned gene is thus functional.     

Analysis of DNA from human adenovirus type 6 with restriction endonucleases HindIII, BglIII and BamHI

The genome of the type 6 human adenovirus has three restriction sites for R.BamHI, thirteen for R.HindIII and ten for R.BglII. The terminal fragments of DNA cleaved by each of the enzymes have been determined by means of terminal nucleotidyl transferase and by analysis of the DNA-terminal protein complex. The sequence of the cleaved fragments has been determined by partial cleavage of DNA, simultaneous digestion of DNA with various combinations of enzymes and secondary digestion of individual isolated fragments with other enzymes. The following order of the cleaved fragments in the adenovirus type 6 genome has been found (the figures in brackets are the weights in mega-daltons): for R.BamHI-B(7.1)-D(3.0)-C(4.05)-A(8.5); for R.HindIII-F(1.7)-C1(2.14)-A(3.44)-M(0.046)-I(1.24)-J(0.77)-D(2.1)-E(1.96)-B(3.18)-H(1.36)-L(0.18)-C2(2.14)-G(1.44)-K(0.16); for R.BglII-E(2.07)-B(3.58)-A(4.8)-C(3.36)-I(0.78)-D(3.25)-G(1.37)-J(0.21)-F(1.85)-K(0.17)-H(0.94).     

Amplification of alpha-fetoprotein complementary DNA by insertion into a bacterial plasmid.

A fragment of the α-fetoprotein (AFP) structural gene was purified and amplified by bacterial cloning techniques. Double-stranded DNA_AFP was constructed from a cDNA copy of greater than 95% pure mRNA_AFP and inserted into E. coli plasmid pBR322 by poly(dA-dT)-linkers. Chimeric plasmid DNA isolated from transformants of E. coli strain χ1776 have been shown to contain α-fetoprotein sequences by hybridization to labeled mRNA_AFP. One clone, designated pA5 (chimeric plasmid pBR322 containing a cDNA_AFP sequence isolated from clone 5), has been studied in more detail. The inserted sequence of approximately 950 nucleotide pairs was positively identified by a hybridization-translation procedure. Hybridization of [³H]uridine-labeled poly(A)-containing RNA from an AFP-secreting cell line to excess pA5 DNA immobilized on nitrocellulose filters was used to show the selectivity of this probe for detecting expression of the AFP gene.     

A Novel Method to Clone P450s with Modified Single-Specific-Primer PCR

We present a method to identify cDNA clones of a cytochrome P450 enzyme. Flavonoid-3', 5'-Hydroxylase (F3',5'H), the key enzyme for the expression of blue or purple color in flowers, was cloned as an example. We have made a catalog of cDNA fragments encoding conserved regions of P450s for petunia (Petunia hybrida Vilm.) petals. Single specific primers were designed for these cDNA sequences and RT-PCRs were performed with cDNA templates. The amplified bands were tested for linkage to the delphinidin producing phenotype using a backcrossed population that had been prepared to have a genetic background of cyanidin-type petunia but segregated for the hydroxylation at the B-ring of anthocyanin. We were successful in amplifying a cDNA fragment that has close linkage to the F3',5'H gene. A full length cDNA clone of the F3',5'H gene was isolated using the amplified fragment as a probe.     

Cloned fragments of human adenovirus type-12 DNA

The following restriction endonuclease fragments of human adenovirus type 12 (Ad12) DNA have been cloned in plasmid or bacteriophage lambda vectors using standard protocols: the EcoRI-A*, -B, -D, -E, and -F fragments, the BamHI-B, -C, -D, -F, -G, -H, and -I fragments, the HindIII-F and -I fragments, and the PstI-A, -D, -F, -G, and -H fragments. The EcoRI-A* fragment comprises the right terminal 5 kb of Ad12 DNA including the terminal 143 bp.