Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.     

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.     

The construction, identification and partial characterization of plasmids containing guinea-pig milk protein complementary DNA sequences.

A complementary DNA (cDNA) plasmid library has been constructed in the plasmid pAT153, using poly(A)-containing RNA isolated from the lactating guinea-pig mammary gland as the starting material. Double stranded cDNA was inserted into the EcoRI site of the plasmid using poly(dA . dT) tails, then transformed into Escherichia coli HB101. From the resulting colonies we have selected and partially characterized plasmids containing cDNA copies of the mRNAs for casein A, casein B, casein C and alpha-lactalbumin. However, the proportion containing casein C cDNA was exceptionally low, and these contained at best 60% of the mRNA sequence.     

Identification and analysis of Dictyostelium actin genes,a family of moderately repeated genes

Plasmid M6 has been shown to contain sequences complementary to two related abundant mRNA species which differ in length by 100 nucleotides and code for Dictyostellum actin. M6 complementary RNA was isolated by hybridization to immobilized M6 DNA and translated in vitro. The product is identical to major forms of in vivo labeled actin in both mobility on two-dimensional gels and two-dimensional fingerprints of tryptic peptides. Both plasmid M6 and a second plasmid complementary to the actin mRNA complementary region in M6, pDd actin 2 (McKeown et al., 1978), direct the synthesis in minicells of a number of similar polypeptides that are not seen in minicells containing other recombinant plasmids. Three of these polypeptides are similar in two-dimensional gel mobility to Dictyostelium actin and bind to DNAase I agarose.The repetition frequency of isolated restriction fragments from actin mRNA complementary plasmid M6 has been examined. The data from two different experimental approaches (DNA excess hybridizations using plasmid DNA as probe, and hybridization of plasmid probe to DNA blot filters of restriction enzyme-digested Dictyostelium DNA) indicate that the mRNA complementary region is reiterated 15–20 times. When an actin cDNA probe is used in the same experiments, the results suggest that the entire coding region is reiterated.When the two major actin mRNA species are separated and independently translated, each appears to code for one of the two major actin species. The results suggest that there are at least two different functional genes, and possibly more, for Dictyostelium actin.     

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.     

Activation of Balbiani ring genes inChironomus tentans after a pilocarpine-induced depletion of the secretory products from the salivary gland lumen

We have constructed recombinant plasmid libraries containing complementary DNA (cDNA) inserts made to poly(A)⁺ RNA isolated from two stages of Dictyostelium development. The procedure utilized for the cloning allows the excision of the cDNA inserts free of vehicle sequences. The two libraries were screened for inserts complementary to moderately abundant and abundant poly(A)⁺ RNA whose genes are differentially modulated during Dictyostelium development. Several of these plasmids were then further examined by hybridization techniques to determine the reiteration frequencies of their genes, the relative rate of complementary RNA synthesis during development, and the relative accumulation and disappearance of complementary RNA during the Dictyostelium life cycle. RNA complementary to two sequences was found to accumulate from approximately one molecule per cell during vegetative growth to several hundred molecules during preaggregation.     

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 amplification of rabbit alpha- and beta-globin gene sequences into Escherichia coli plasmids.

cDNA, synthesized from rabbit globin mRNA, was used in a self-priming reaction, with avian myeloblastosis virus DNA polymerase, for the synthesis of double-stranded DNA. Globin DNA ranging from about 400 to 650 base pairs was elongated with dG tails using deoxypolynucleotide transferase and was annealed to linear Escherichia coli plasmic pCR1, elongated with dC tails. Preparation of the plasmid DNA involved an enzymatic reconstruction of one EcoRI-specific site on each side of the molecule. After transformation of E. coli cells to kanamycin resistance with the hybrid molecules, bacterial clones harboring recombinant plasmids were studied for the presence of globin-specific DNA. Plasmids containing either alpha or beta rabbit globin gene sequences were obtained. There was a 4-fold excess of recombinant plasmids containing beta-globin sequences over those with alpha-globin DNA. The longest beta-globin sequences found in plasmids were about 550 to 600 pairs long, and correspond therefore to the entire beta-globin structural gene and to some of the untranslated regions. The alpha-globin sequences were 400 to 450 base pairs long. Treatment of clone pCR1betarG 19 with EcoRI endonuclease released two DNA fragments (410 and 210 base pairs) resulting from cleavage at two reconstructed external EcoRI sites and at one internal EcoRI site within the rabbit globin gene. The same treatment of pCR1alpharG 11 released one fragment. In most other recombinant plasmids studied however, no fragment was released by EcoRI digestion.     

Characterisation of bacterial clones containing DNA sequences derived from Xenopus laevis vitellogenin mRNA.

A 1700 nucleotide DNA sequence derived from Xenopus vitellogenin mRNA has been cloned in the bacterial plasmid pBR322. The identity of the cloned sequence was verified in two ways. Firstly, the plasmid DNA was shown to hybridise to an RNA of the correct size (6,700 nucleotides). This was shown by in situ hybridisation to electrophoretically separated RNA and also by the formation of "R-loops" with purified vitellogenin mRNA. Then, using a novel procedure in which plasmid DNA covalently bound to diazotised paper is used to select complementary mRNA sequences, the cloned sequence was shown to hybridise to an mRNA which directed the synthesis of vitellogenin when translated in a reticulocyte lysate cell-free system.     

Characterization of a serine carboxypeptidase in the salivary glands and fat body of the orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae)

A full-length cDNA encoding a serine carboxypeptidase (designated SmSCP-1) was recovered from an ongoing salivary gland EST project of the wheat midge. The deduced 461-amino acid sequence had a putative signal sequence at the amino terminus, indicating it was a secreted protein. The protein shared homology with serine carboxypeptidases from other insects, mammals, plants, and yeasts. SmSCP-1 mRNA was expressed in all stages of development and detected in salivary gland and fat body tissues but not in midgut tissue. Expression analysis and quantitative real-time PCR assays in male and female wheat midges and the fat body tissue of adult midges revealed that SmSCP-1 was up-regulated nearly four-fold in the female midges compared to males and nearly two-fold in female fat body compared to male fat body. The wheat midge serine carboxypeptidase (SmSCP-1) most likely has a dual function. As a secreted digestive enzyme, it could play a role in mobilizing host-plant seed reserves for feeding larvae and as expressed in fat body could function as an exopeptidase in degradation of vitellogenin and/or in post-translational processing of other enzymes.