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.     

High-efficiency cloning of full-length cDNA.

A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.     

Molecular cloning and sequence analysis of full-length cDNA for mRNA of adrenodoxin oxidoreductase from bovine adrenal cortex

A full-length cDNA clone (pADR) for adrenodoxin reductase was isolated by means of immunological screening from a bovine adrenal poly(A)+ mRNA library. A cDNA insert of 1,973 base pairs in length encoded the entire amino acid sequence of the adrenodoxin reductase precursor protein, which consists of 492 amino acids including an extrapeptide of 32 amino acids at the NH2-terminus. The cloned cDNA contained the complete 3'-noncoding region of 443 nucleotides including 59 nucleotides of poly(A) and 51 nucleotides in the 5'-noncoding region. The amino acid sequences from the 33rd to 70th, the 117th to 123rd, the 207th to 225th, the 247th to 323rd, the 385th to 426th, the 444th to 461st, and the 487th to 492nd in the predicted structure were identical with those of the purified adrenodoxin reductase and its digested peptides, with only four exceptions.     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

cDNA cloning of putative rat acetyl-CoA transporter and its expression pattern in brain

Rat acetyl-CoA transporter gene (Acatn) encodes a hydrophobic multi-transmembrane protein involved in the O-acetylation of gangliosides. O-acetylated gangliosides have been found to play important roles in the embryonic development of the nervous system. We have isolated rat Acatn cDNA by PCR cloning. The amino acid sequence of rat Acatn exhibited 92% and 96% homology with human and mouse sequences, respectively. The mRNA was expressed in brain at all developmental stages. Acatn expression was higher in embryonic and postnatal rats than in adult rats. Cellular localization of Acatn mRNA in adult rat brain was also analyzed by in situ hybridization. Acatn mRNA expression was detected in the neuronal cells of cerebellum, hippocampus, hypothalamus, cortex, olfactory bulb, and dorsal and ventral anterior olfactory nucleus in adult rat brain.     

Molecular cloning and sequence analysis of full-length cDNA coding for mouse contrapsin

Four overlapping cDNA clones encoding contrapsin were isolated from a mouse liver cDNA library constructed in the expression vector, lambda gt11. M13 vector sequence analysis revealed that contrapsin cDNA contained an open reading frame of 1,254 bases encoding 418 amino acids. The N-terminal amino acid sequence of the isolated contrapsin matched residues 30 to 48 of the sequence deduced on nucleotide analysis. One clone, which had the longest 3' untranslated region, contained two sets of tandem polyadenylation signals, AATACA and AATAAA, which were located 497 bases apart, while the remaining three clones terminated at the first signal. The entire reading frame sequence of contrapsin cDNA showed 64% homology with that of human alpha-1-antichymotrypsin.     

Molecular cloning and characterization of a full-length cDNA clone for human plasminogen

A human liver cDNA library enriched for full-length clones was screened for plasminogen cDNA using a synthetic 24-nucleotide probe derived from a reported partial cDNA sequence. 12 positive clones were identified and one of these was characterized in detail. The 2.7 kb insert contains the complete coding region. At 5 positions, it gives residues different from those reported in a previous amino acid sequence analysis of the protein. The present results show an extra Ile at position 65, Gln instead of Glu at positions 53 and 342, Asn at position 88 instead of Asp, and Asp at position 453 rather than Asn. In the 3'-non-coding region an extension of 29 bases is found which does not contain any structure compatible with a known polyadenylation signal. Instead, the consensus signal AATAAA is placed at a distance of 46 bases upstream of the poly(A)-tail.     

“电子”cDNA文库筛选指导基因的全长cDNA克隆

“电子”ｃＤＮＡ库筛选主要是指通过采用生物信息学的方法延伸表达序列标签（ＥＳＴ）序列,以获得基因的部分及至全长ｃＤＮＡ序列,避免或部分避免构建与筛选ｃＤＮＡ库等烦琐的实验室工作。该方法具体体现了ＥＳＴ数据库的迅速扩张已导致识别与克隆新基因的策略发生革命性的变化。ＥＳＴ序列ＺＡ７３为本实验室克隆到的可能参与辐射致气管上皮细胞恶性转化过程的基因片段,本研究采用“电子”ｃＤＮＡ库筛选的方法对其可能     

cDNA cloning and characterization of vinculin mRNA

Vinculin and meta-vinculin are related proteins that localize to adherens junctions. To facilitate studies on the biology of these proteins cDNA clones were isolated from a chick embryonic library made in lambda gt 11. Three clones, accounting for 1/2 of the vinculin message, were identified by immunological analyses of fusion proteins and by hybrid-arrested translation. Sequence analyses of the 3' ends of these clones reveal allelic variants. Northern blot analyses suggests the presence of multiple vinculin messages, the most prominent one at 6.2 kb, almost twice the size needed to encode vinculin.     

A specific human lysophospholipase: cDNA cloning, tissue distribution and kinetic characterization

Lysophospholipases are critical enzymes that act on biological membranes to regulate the multifunctional lysophospholipids; increased levels of lysophospholipids are associated with a host of diseases. Herein we report the cDNA cloning of a human brain 25 kDa lysophospholipid-specific lysophospholipase (hLysoPLA). The enzyme (at both mRNA and protein levels) is widely distributed in tissues, but with quite different abundances. The hLysoPLA hydrolyzes lysophosphatidylcholine in both monomeric and micellar forms, and exhibits apparent cooperativity and surface dilution kinetics, but not interfacial activation. Detailed kinetic analysis indicates that the hLysoPLA binds first to the micellar surface and then to the substrate presented on the surface. The kinetic parameters associated with this surface dilution kinetic model are reported, and it is concluded that hLysoPLA has a single substrate binding site and a surface recognition site. The apparent cooperativity observed is likely due to the change of substrate presentation. In contrast to many non-specific lipolytic enzymes that exhibit lysophospholipase activity, hLysoPLA hydrolyzes only lysophospholipids and has no other significant enzymatic activity. Of special interest, hLysoPLA does not act on plasmenylcholine. Of the several inhibitors tested, only methyl arachidonyl fluorophosphonate (MAFP) potently and irreversibly inhibits the enzymatic activity. The inhibition by MAFP is consistent with the catalytic mechanism proposed for the enzyme - a serine hydrolase with a catalytic triad composed of Ser-119, Asp-174 and His-208.     

High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper

Full-length cDNAs are essential for functional analysis of plant genes. We constructed high-content, full-length cDNA libraries from Arabidopsis thaliana plants based on chemical introduction of a biotin group into the diol residue of the CAP structure of eukaryotic mRNA, followed by RNase I treatment, to select full-length cDNA. More than 90% of the total clones obtained were of full length; recombinant clones were obtained with high efficiency (2.2 × 10⁶/9 μg starting mRNA). Sequence analysis of 111 randomly picked clones indicated that 32 isolated cDNA groups were derived from novel genes in the A. thaliana genome.     

Efficient cloning of full-length cDNAs based on cDNA size fractionation

The ability to generate and obtain full-length (FL) cDNAs is of critical importance to the field of genomics. Most cDNAs in a traditional cDNA library lack the initiating 5' ATG, making it difficult to obtain a FL clone. We report here on an improved protocol for the preparation of FL enriched cDNA libraries. We demonstrate that if good quality RNA is used in the cDNA synthesis, high-quality, FL cDNA can be generated for messages upward of 7 kb. In addition, we demonstrate the utility of size fractionation as a means to produce libraries containing a high percentage of initiating 5' ATG containing clones with insert sizes greater than 4 kb. The method is simple, cost efficient, and can be performed in most laboratories equipped to perform molecular biology. Lastly, the novel methodologies used in the analysis of the cDNA and library should prove useful to others working to create high-quality cDNA libraries.     

cDNA cloning and structure of mouse putative Ah receptor.

Mouse cDNA clones for a putative Ah receptor have been isolated from a cDNA library of mRNA from Hepa-1 cells by an oligonucleotide probe produced by PCR with a pair of primers which was synthesized according to the reported N-terminal sequence of 26 amino acids. The cDNA clones encode a polypeptide of 805 amino acids with a helix-loop-helix motif and with some similarity to a certain region designated PAS of Drosophila Per and Sim, and human Arnt protein. Cotransfection of an expression vector of the Ah receptor with a reporter plasmid pMC6.3k consisting of CYP1A1 promoter and CAT structural gene into CV-1 cells enhanced the CAT expression in response to added 3-methylcholanthrene.     

Molecular cloning of a full-length cDNA for human alpha-N-acetylgalactosaminidase (alpha-galactosidase B)

In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality alpha-N-acetylgalactosaminidase. The full length cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human alpha-galactosidase A and yeast alpha-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality alpha-N-acetylgalactosaminidase.     

cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA.

We report the cloning of a full-length cDNA encoding rat preproendothelin-1 (preproET-1). The predicted rat preproET-1 consists of 202 amino acid residues and highly similar to human, porcine and bovine preproET-1, respectively. The deduced 21-residue sequence of mature rat ET-1 is identical to human, porcine, canine and bovine ET-1. As in other mammalian species, the mature ET-1 is predicted to be produced from a 39-residue big ET-1 in the rat. Northern blot analysis showed that a single 2.3-kb preproET-1 mRNA is expressed not only in vascular endothelial cells but also in other rat tissues, including the lung, brain, uterus, stomach, heart, adrenal gland and kidney. These findings suggest that ET-1 may play roles as a local mediator in multiple organs both within and outside the cardiovascular system in the rat.