Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Structure of mouse interferon-beta cDNA clones: sequence rearrangements in a cloned cDNA

A fraction enriched in interferon (IFN) mRNA was prepared from mouse C243-3 induced cells and was used for the construction of a cDNA library. Two plasmids were obtained after screening by differential colony hybridization and IFN mRNA hybridization-selection and translation. The nucleotide sequences of the cDNA inserts revealed that both were partial copies of IFN-beta mRNA. The cDNA 861 corresponds to the entire 3' nontranslated region of the mRNA while the cDNA 2939 consists of rearranged translated regions of IFN mRNA. A mechanism for the rearrangement events during cDNA synthesis is proposed. A chromosomal DNA fragment hybridizing to cDNA 2939 was identified by screening a mouse genomic library.     

Rat aldolase isozyme gene

Rat aldolase B mRNA was partially purified from liver polysomes by an immunochemical technique followed by oligo(dT)-cellulose column chromatography. Double-stranded cDNA, synthesized from this mRNA, was inserted into the PstI site of plasmid pBR322 employing the oligo(dC)-oligo(dG) tailing method. Clones containing aldolase B cDNA inserts were selected by colony hybridization using 32P-labeled purified mRNA as a specific probe. Several recombinant plasmids containing 600 to 1000 base pair inserts were isolated. Hybrid selection-translation experiments showed that they hybridize specifically with aldolase B mRNA. By overlapping restriction maps of several individual cDNA inserts, it was found that they spanned 1200 base pairs, which represented about 70% of the aldolase B mRNA sequence. The nucleotide sequence of the cDNA was then determined and the sequence of 180 amino acids from the COOH terminus and the entire 3' untranslatable nucleotide sequence were clarified. Although the complete amino acid sequence of rat aldolase B has not yet been reported, it was found that several amino acids neighboring the COOH-terminal tyrosine obtained by carboxypeptidase digestion completely coincided with those determined from the cDNA sequence; i.e. -Ser-Leu-Phe-Thr-Ala-Ser-Tyr-Thr-Tyr. Furthermore, a putative active site peptide appeared and is extensively homologous to those of rabbit aldolases A and B.     

Molecular cloning of cDNA for rat acyl-CoA oxidase

Poly(A+) RNA was prepared from hepatic free polysomes of rats which had been fed di(2-ethylhexyl) phthalate for the induction of peroxisomal beta-oxidation enzymes. This preparation was enriched for the mRNAs of these enzymes by sucrose density gradient centrifugation, and used for the synthesis of double-stranded cDNA. Recombinant plasmids were constructed from the cDNA and pBR322 by dG X dC-tailing method and used for the transformation of an Escherichia coli strain, chi 1776. By differential colony hybridization using [32P]cDNA of partially purified liver poly(A+) RNA from induced and noninduced rats as probes, and then by hybridization-selected translation, we obtained two clones with cDNA inserts which specifically selected acyl-CoA oxidase mRNA. On Northern blotting, both cDNA inserts hybridized to 3.8-kilobase RNA which was increased about 10-fold by di(2-ethylhexyl) phthalate treatment of the rats. The cleavage maps of the cDNA inserts showed they overlap with each other. We conclude that the above two recombinant plasmid clones contain cDNA sequences for rat acyl-CoA oxidase.     

Molecular cloning of partial cDNA copies of two distinct mouse IFN-beta mRNAs.

Two cDNA libraries were constructed, using respectively the 12S and the 16S sucrose gradient fractions of polysomal poly (A)+ RNA from mouse C243 cells induced with Newcastle disease virus. Screening of a part of both libraries by mRNA selection hybridization assays revealed the presence of two plasmids hybridizing to an mRNA, whose translation product was characterized as mouse IFN-beta. Blot analysis of RNA indicated that mRNA hybridizing to the DNA from both plasmids could be detected in induced but not in uninduced C243 cells. The two cDNA inserts did not cross hybridize and had distinct restriction maps. Sequencing revealed that both inserts represented the end of the coding region and the entire 3' non coding region of two district mRNAs. Although different, the putative 39 AA and 65 AA carboxy termini of both Mu IFN-beta s display some homology to human IFN-beta 1. Thus there are at least two different murine IFN-beta genes.     

Molecular cloning of cDNA for rat and human carbamyl phosphate synthetase I

Recombinant plasmids with inserts complementary to the mRNA for carbamyl phosphate synthetase I were identified from a rat liver cDNA library by hybrid-selected mRNA translation. Four clones, the largest being 3100 base pairs, were identified for the rat liver enzyme. Using the rat liver cDNA as a probe, two homologous recombinant plasmids of approximately 1200 base pairs in length were isolated from a human liver cDNA library. Northern blot analysis of rat liver mRNA and baboon liver mRNA revealed the presence of a 5000-base mRNA homologous to both rat and human cDNA probes. No homologous mRNA was observed in mRNA from rat heart or rat kidney as is consistent with the known tissue distribution of this enzyme. The induction of carbamyl phosphate synthetase and argininosuccinate synthetase mRNA during the fetal and postnatal development of the rat was studied by dot blot analysis of isolated mRNA. The mRNA for both enzymes appeared between 17 and 19 days of fetal life and reached approximately 40% of adult levels during this period. This initial increase was followed by a rapid decline just prior to birth. The mRNA levels slowly increased during postnatal life, not reaching adult levels until after the 20th day of neonatal life. Using the human cDNA clones, the human carbamyl phosphate synthetase gene was mapped to chromosome 2 utilizing a panel of Chinese hamster X human somatic cell hybrids. Analysis of one hybrid with a human-Chinese hamster translocation provided a provisional assignment to the short arm of chromosome 2.     

Alkaline phosphatase conjugated protein A as a sensitive reagent to immunoscreen an expression cDNA plasmid library: isolation of cDNA to the calcium-binding protein of the chick embryonic chorioallantoic membrane

A highly efficient immunoscreening procedure has been developed to isolate cDNA clones to the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM). A library of total CAM cDNA was constructed using the expression plasmid vector, pUC 19. Bacterial clones containing plasmids with CaBP cDNA inserts were detected immunohistochemically based on their expression of hybrid CaBP protein sequences. For immunodetection, nitrocellulose bacterial colony replicas were treated with specific antibodies to the CaBP followed by incubation with Staphylococcus aureus Protein A conjugated with alkaline phosphatase (AP) which served as a secondary immunoreagent. Positive clones were then histochemically identified based on AP enzyme activity. The identity of the immunopositive clones was further verified by in vitro translation of mRNA selected by hybridization to the cloned cDNA. The AP-based immunoscreening procedure yields stable reaction products with relatively low background, and should find general application for isolating specific cDNA clones from expression cDNA libraries.     

Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis.

We have developed a new class of cloning vectors: lambda-full-length cDNA (lambda-FLC) cloning vectors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, lambda-FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain "size balancers." Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre-lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors.     

Cloning of a cDNA complementary to rat preputial gland β-glucuronidase mRNA

Messenger RNA was isolated from rat preputial glands by guanidine HCl extraction, ethanol and salt precipitation, followed by chromatography on oligo(dT) cellulose. Double-stranded cDNA was synthesized from the mRNA and inserted into the Pst 1 site of the plasmid pBR322 by the poly(dG)·poly(dC) tailing and annealing procedure. The hybrid plasmids were used to transform $\text{E. coli}$ HB101. Recombinant clones were screened for those containing cDNA inserts complementary to β-glucuronidase mRNA by a hybridization-selection procedure. One clone, containing an insert of about 1.2 kilobases, hybridized to preputial gland mRNA which, when translated $\text{in vitro}$, gave a product that migrated with the β-glucuronidase subunit on polyacrylamide gels.     

Isolation of cDNA for Pea Phytochrome Using an Expression Vector

Partially purified phytochrome mRNA was obtained from etiolatedpea epicotyls by polyribosome immunoprecipitation or by sizefractionation of total poly(A)⁺RNA, and used for the synthesisof double-stranded complementary DNA (cDNA). cDNA librarieswere constructed using an Escherichia coli expression vector,pUC9, and screened for phytochrome cDNA by colony immunologicalassay. Nine colonies were found to produce a 27 kDa polypeptidethat was reactive to both polyclonal and monoclonal antipeaphytochrome antibodies. The plasmids from these colonies containedcDNA inserts of 1.2 or 2.0 kbp. Hybridization-arrest translationassay verified that the cDNA clones contained a sequence codingfor phytochrome polypeptide. RNA blot hybridization analysisindicated that the cDNA hybridized to a 4.1 kb poly(A)⁺RNA indark-grown pea. (Received March 22, 1986; Accepted June 13, 1986)     

Molecular Cloning and Characterization of Stored mRNA in Cotyledons of Vigna unguiculata

From a gt1O cDNA library constructed from the total polyadenylatedRNA (poly A⁺ RNA) of mature cowpea cotyledons, we selected recombinantphages that hybridized with a cDNA probe complementary to polyA⁺ RNA from cotyledons collected 24 h after the onset of imbibition(cDNA-B) but that did not hybridize with a probe from cotyledonsat developmental stage II (13 to 15 days after flowering; cDNA-A).cDNA inserts of two of these phages were subcloned in pUC18plasmids and the resultant plasmids were designated pSAS5 andpSAS1O, respectively. We also selected two recombinant phagesthat hybridized with cDNA-A but not with cDNA-B, and one ofthem was subcloned in pUC18 to generate pSASC1. We then characterizedthe pSASlO cDNA insert as the cDNA of a putative stored mRNAof cowpea seeds and the pSASC1 insert as a reference cDNA. TheRNA-blot hybridization after gel electrophoresis, with pSAS10as probe, indicated that an essentially full-length cDNA hadbeen cloned, and the hybrid-select translation of pSAS10 mRNAgave a 10-kDa product. pSASC1 mRNA was shown to code for a 46-kDapolypeptide, which corresponds in size to one of the major storageproteins of the cowpea. pSAS10 mRNA was detectable only in cotyledonsat developmental stage III (17 to 19 days after flowering) orlater, and the level of the mRNA began to decline when seedsgerminated. The mRNA was also present in cowpea embryonic axes.mRNA hybridizable to pSAS10 cDNA was found in extracts of someother plants. (Received July 17, 1989; Accepted October 17, 1989)     

Rapid quantitation of individual RNA species in a complex population.

Many investigations require quantitation of one or more individual RNA species in complex populations. Existing methods are tedious when multiple samples are to be assayed. A method is presented which allows rapid and accurate quantitation of many species of RNA simultaneously. Recombinant plasmids containing cDNA inserts are electrophoresed in agarose and blotted to nitrocellulose. After hybridization with labeled RNA and autoradiography, bands are quantitated by scanning. The results were calibrated by solution hybridization. The approach has been validated through the use of plasmids containing inserts of Drosophila cDNA and RNA of cultured cells.     

Identification of clones containing DNA complementary to phenylethanolamine N-methyltransferase mRNA

Specific poly(A)mRNA for phenylethanolamine N-methyltransferase was isolated from bovine adrenal medulla by immunoprecipitation of polysomal mRNA with antibodies to bovine adrenal phenylethanolamine N-methyltransferase. Antibody-polysome complexes were recovered by Protein A Sepharose affinity chromatography. Phenylethanolamine N-methyltransferase mRNA, enriched 50-fold as judged by quantitative immunoprecipitation of translation products, was used as a template for the synthesis of complementary DNA (cDNA). Double-stranded cDNA was tailed with deoxycytosine and inserted into the Pst 1 site of poly(dG)-tailed plasmid pBR322. The resultant recombinant plasmids were used to transform competent E. coli strain 294. Tetracycline-resistant ampicillin-sensitive clones were screened by positive hybridization selection, and preliminary screening identified 2 out of 36 clones containing phenylethanolamine N-methyltransferase cDNA inserts. One phenylethanolamine N-methyltransferase cDNA insert was isolated from the plasmid DNA by digestion with Pst 1 and was found to be approximately 350 base-pairs in length. Northern blot analysis revealed that this phenylethanolamine N-methyltransferase cDNA probe strongly hybridized to an RNA species of approximately 1100 nucleotides.     

Cloning and characterization of synthetic sequences from the Xenopus laevis vitellogenin structural gene

The mRNA coding for vitellogenin, the yolk protein precursor, has been isolated from the liver of estrogen-stimulated Xenopus laevis. The mRNA has a size of 6.3 kilobases (kb). Optimal conditions were investigated for the synthesis of long complementary DNA (cDNA, referring to DNA synthesized in vitro) copies of the mRNA. Temperature, salt concentration, and enzyme-to-RNA ratio were important factors. Double-stranded cDNA with an average size of 2 to 3 kb was inserted into the vector pMB9 by the poly(dA:dT) method, and the recombinant plasmids were amplified in E. coli. Twenty-one clones with vitellogenin inserts ranging from 1 to 3.7 kb were studied. The regions in the RNA from which these clones had been derived were mapped by R-loop analysis in the electron microscope and by hybridization of the cloned DNAs with specific fractions of mRNA. Slightly more than half of the clones were derived from the 3′-terminal portions of the mRNA while the remaining clones are located internally.     

The complete sequence of human preprocalcitonin

DNA complementary to mRNA extracted from the thyroid glands of patients suffering from medullary carcinoma of the thyroid (MCT), a calcitonin-producing tumour, was inserted in the Pst site of pBR 322 by G-C tailing. The recombinant plasmids were used to transform Escherichia coli DP 50. Ampicillin-resistant clones were screened using a 32P-labelled cDNA to mRNA extracted from a case of MCT particularly rich in calcitonin (CT) mRNA. Positive clones were subsequently rescreened using a 32P poly(T) probe. Eighty clones were thus purified, and the inserts obtained by digestion with PstI were subjected to positive hybridization selection with subsequent translation in vitro. An insert stimulating synthesis of the protein and containing restriction sites compatible with the previously published complete sequence of calcitonin mRNA from rat was sequenced. This cDNA insert contained the entire coding region of 426 bp, 70 bp at the 5'-end, and 295 bp upstream from the poly(A) tail. The complete amino acid sequence of human preprocalcitonin could thus be deduced.     

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

A rapid method for cloning and sequencing variable-region genes of expressed immunoglobulins

A rapid procedure is described for cloning immunoglobulin V region genes from cells that express them. cDNA is synthesized from mRNA template using primers homologous to the immunoglobulin constant-region genes. Blunt-ended, double-stranded cDNA is obtained by sequential addition of enzymes to a single tube. The cDNA is inserted directly into the M13 vector, which is screened by plaque lifting for the presence of specific inserts. Screening probes can be generated from 32P-labeled single-stranded cDNAs generated from primers different from those used for cloning, or alternatively, from previously cloned V or C gene segments. The ease of cloning a cDNA V region is directly related to the abundance of Ig-specific mRNA within the cell of interest. This method minimizes the number of steps and the time needed to obtain accurate and complete sequences of any expressed Ig V region gene.