5''-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency.

A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5'-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the S1 nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3'-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3'-tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5'-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences. Chicken lysozyme cDNA was cloned into pBR322 by this procedure with a transformation efficiency of 5 x 10(3) recombinant clones per ng of ds-cDNA. Sequence analysis revealed that at least nine out of nineteen randomly isolated plasmids contained the entire 5'-untranslated mRNA sequence. The data strongly support the conclusion that the 5'-untranslated region of the lysozyme mRNA is heterogeneous in length.     

Cloning of Protamine cDNA of the Medaka (Oryzias latipes) and Its Expression during Spermatogenesis

Protamines or sperm specific basic proteins are highly basic low molecular weight proteins that substitute histones in the chromatin of sperm during spermatogenesis. They condense sperm DNA into a highly compact, stable and inactive complex. In this study, cDNA of protamine of the medaka, Oryzias latipes , was cloned to elucidate the molecular mechanisms involved in spermatogenesis. A medaka testis cDNA library constructed in lambda gt 11 showed 2.78X10⁶ independent recombinants. Several positive clones were obtained by immunoscreening with polyclonal antiserum against medaka protamine. Sequencing showed that one of these positive clones, named MP-1, encoded arginine clusters characteristic of protamine. The putative amino acid sequence of MP-1 revealed a remarkable extent of homology with other fish protamines, such as 71% identity with thynnin Y, a sperm specific basic protein isolated from the bluefin tuna, Thunnus thynnus . Northern hybridization using a MP-1 cDNA probe showed that MP-1 mRNA is present exclusively in the testes and that it gave three detectable bands: a major band of 280 b, and two others of 400 b and 500 b. In situ hybridization of a complementary RNA probe (digoxigenine-UTP-labeled MP-1 RNA) revealed that MP-1 mRNA is localized in some secondary spermatocytes and spermatids, but not in primary spermatocytes or spermatogonia. These results differ from those obtained in studies on the rainbow trout by solution hybridization and in situ hybridization.     

Isolation of non-globin genes expressed preferentially in mouse erythroid cells.

Genomic DNA recombinants were isolated from a library of Balb-C mouse genomic DNA fragments cloned in lambda Ch4A by screening with cDNA derived from 13d foetal liver cell or adult reticulocyte poly A+ RNA. Subsequent screening enabled us to identify non-globin genomic sequences whose expression appeared exclusive to or elevated in erythroid cells. Further analysis of the structure and expression of these sequences was performed using Southern blot and DNA or RNA dot hybridisation analysis. In one recombinant part of the cloned genomic sequence corresponded to an erythroblast specific mRNA identified previously by Affara et al, (5).     

Nucleotide sequence of a protamine component CII gene of Salmo gairdnerii.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".     

Protamine messenger RNA: evidence for early synthesis and accumulation during spermatogenesis in rainbow trout

Trout testis cells were separated into various developmental classes by velocity sedimentation in bovine serum albumin gradients and were identified morphologically with particular stages of the process of spermatogenesis. The stage of testis cell differentiation at which protamine mRNA appears in the cell cytoplasm for the first time was determined by hybridization of RNA populations extracted from the separated cells to radioactively labeled protamine cDNA. Primary spermatocytes represent the earliest stage of differentiation at which protamine mRNA can be detected in large quantities in the cell cytoplasm, establishing that the synthesis of this class of mRNA occurs at a much earlier stage than the time of its translation at the spermatid stage. Protamine mRNA sequences were found in both the polysomes and postribosomal supernatant of the spermatid cells which are involved in the synthesis of protamine, while primary and secondary spermatocytes contained the mRNA sequences only in their postribosomal supernatant fractions. These findings strongly suggest that protamine mRNA is synthesized, accumulated, and stored in the cell sap of primary and secondary spermatocytes in the form of “inactive” messenger ribonucleoprotein particles, which are “activated” and translated at the spermatid stage.     

Use of matrix-immobilised recombinant plasmids to purify chain-specific rabbit globin complementary DNAs.

The cloning of DNA sequences in plasmid recombinants has made it possible to amplify specific sequences to an extent that they can be used for preparative purposes. We describe the use of rabbit globin DNA sequences cloned in the plasmid pCR1 and covalently bound to Sepharose 4B for the purification of chain-specific rabbit alpha- and beta-globin cDNAs. These purified probes were then used to estimate the length of the alpha- and beta-globin DNA sequences inserted into the recombinant plasmid. The technique should allow the rapid isolation of sequence-specific cDNA, RNA and genomic DNA.     

A single receptor identical with that from intestine/T47D cells mediates the action of 1,25-dihydroxyvitamin D-3 in HL-60 cells.

Two anti-vitamin D receptor monoclonal antibodies binding to two different epitopes immunoprecipitate 100% of the HL-60 1,25-dihydroxyvitamin D-3 binding activity, while another monoclonal antibody specific for the porcine receptor precipitates none. Using a rat receptor cDNA probe, a single mRNA species of 4.6 kb was detected by Northern analysis of HL-60 mRNA. Using a cDNA probe from the cloned rat receptor, 10(7) recombinants from a lambda gt11 cDNA library constructed from mRNA isolated from HL-60 cells was screened yielding two positive clones. These clones had sequences identical with the known human receptor sequence from intestinal/T47D sources. Using PCR technology, the entire sequence of the HL-60 1,25-dihydroxyvitamin D-3 receptor was determined. This sequence was found identical with that reported for the human intestinal/T47D cDNA encoding the vitamin D receptor except for a single base. The substitution of this particular base does not alter the amino acid sequence however. Thus, the same receptor likely operates in differentiation and calcium transport functions.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Cloning of cDNA to rat mammary-gland fatty acid synthase mRNA. Evidence for the expression of two mRNA species during lactation.

A cDNA library was constructed in the expression vector lambda gt11, by synthesizing cDNA from size-selected poly(A) RNA from lactating rat mammary gland, using random hexanucleotide primers. Using this library we identified two recombinants which, on addition of a lac z inducer, produced proteins recognized by affinity-purified anti-fatty-acid synthase antibody, and which, therefore, contained fatty acid synthase coding sequences. The inserts were subcloned, were shown to be between 500 and 600 base pairs in size, and to cross-hybridize. The cloned DNA was then used in Northern hybridizations with mRNA isolated at various stages throughout lactation. Two mRNA species were identified of approx. 9.7 and 10.4 kilobases, which increased and decreased in parallel during lactation, reaching a peak at 12-13 days. Both mRNA species disappeared rapidly if the pups were removed prematurely. This study provides evidence that, during hormonal induction in lactation, regulation of the level of fatty acid synthase protein can be accounted for by variation in the level of mRNA.     

Chicken histone H5: selection of a cDNA recombinant using an extended synthetic primer

We describe the use of a synthetic primer to select a cDNA recombinant clone containing H5 coding sequences. The strategy used was as follows: 1. Prepare oligo(dT) cellulose-bound mRNA from chicken reticulocytes and select 11S-18S material from sucrose gradients. 2. Use this RNA fraction both to prepare a cDNA library and as a template for H5-specific cDNA synthesis using a synthetic primer. 3. Screen out most globin cDNA recombinants with oligo(dT)-primed globin cDNA. 4. Search for H5 recombinants using H5 specific cDNA and verify the identity by DNA sequencing. Our screening suggests an H5 mRNA abundance of about two parts per thousand in chicken reticulocyte poly(A)-containing RNA. The isolation of an H5 cDNA recombinant clone is an initial step in the study of H5 genes and their relationship to H1 and core histone genes.     

Nucleotide sequence analysis of the cloned salmon preproinsulin cDNA

A cDNA library was constructed using polyadenylated RNA from salmon (Oncorhynchus keta) Brockmann bodies, plasmid vector pBR322, and in vitro recombinant DNA techniques. Insulin-related clones were identified with a cDNA probe generated from the same RNA and enriched for insulin sequences. Two recombinants were shown to contain the nucleotide sequence of the entire coding region and parts of the 5' and 3' untranslated regions. The salmon preproinsulin mRNA is about 760 nucleotides long, 315 of which code for the protein, while about 190 and 200 nucleotides belong to the 5' and 3' flanking regions, respectively. Comparison of the nucleotide sequences of salmon insulin mRNA with those from other species reveals that sequence conservation is limited to the regions coding for the B and A peptides and two segments of the signal peptide. The C-peptide region exhibits no significant sequence homology with the C-peptides of other vertebrates. The 5' and 3' untranslated regions of the salmon preproinsulin mRNA are homologous only with the anglerfish mRNA, whereas there is no evident homology with those of birds and mammals. In addition to establishing the sequence of the preproinsulin mRNA, cloned salmon insulin cDNA provides a specific probe for the analysis and isolation of genomic DNA fragments containing insulin genes.     

Divergence of protamine gene sequences in fish

Complementary DNA to trout protamine mRNA was hybridized to excess genomic DNA from trout, salmon and yellow perch. Although there was extensive hybridization of the cDNA to trout DNA, no cross-reaction with yellow perch DNA was observed and the hybridization to salmon DNA was noticeably less than in the homologous reaction. To confirm these results, yellow perch protamine mRNA was purified and compared directly to trout protamine mRNA. Yellow perch protamine mRNA was shorter than trout protamine mRNA, when measured by agarose gel electrophoresis in the presence of methyl mercury hydroxide. The two mRNAs did not cross-react in cDNA/RNA hybridizations, although the homologous reactions went to 90% of completion. This lack of sequence homology was confirmed when the oligopyrimidine tracts from the cDNAs were compared. No sequences longer than tetranucleotides were common to both species. Trout protamine cDNA contained oligopyrimidines of composition C₇T₄, C₄T₂, C₃T₂, C₂T₄, C₂T₃, C₁T₅ and C₁T whereas yellow perch protamine cDNA contained C₆T₃ and C₄T₃.     

A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA

In this report we present a strategy for generating a representative cDNA library from prohibitively low amounts of mRNA template. A defined DNA adapter, which carries an EcoRI site, is ligated to both ends of the products of a cDNA synthesis reaction. This allows low levels of cDNA to be amplified by a polymerase chain reaction. In studies with pg amounts of rabbit globin mRNA, the amplified cDNA product is shown to be full-length. Globin cDNA recombinants are positively identified in lambda gt10. The protocol should be widely applicable to mRNAs of low abundance, whose sequences have not been determined, and to limited samples from patients or animals. It may also be useful for generating representative libraries of low titer or variant viral sequences.     

A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein

An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.     

Molecular analysis of the protamine multi-gene family in rainbow trout testis.

We have synthesized a family of double-stranded cDNAs (ds cDNAs) using as a template the family of highly purified protamine mRNAs from rainbow trout testis. Individual pure protamine cDNA components were isolated by cloning this family of protamine ds cDNAs in a plasmid vector (pMB9). Clones containing protamine sequences were characterized by restriction mapping and by a positive hybrid-selected translation assay, which allowed us to correlate particular cDNAs with particular protein components. To allow more detailed comparisons, complete nucleotide sequences were determined for selected protamine clones. We have detected at least 5 distinctly different coding sequences, which nevertheless show at least 82% homology, and which have probably arisen by repeated gene duplication. These very highly conserved coding sequences do however contain a distinctly variable region near the 5'-end of the mRNA (N-terminus of the protein), corresponding to the major sites of serine phosphorylation. Since the amino acid sequences predicted by our DNA sequences were slightly different from those previously published (1), we have independently determined the amino acid sequences of protamine components CI, CII, CIII from our own source of trout testis. These new peptide sequences are completely consistent with those predicted by our nucleotide sequences. The 3'-untranslated regions of the protamine mRNAs are, surprisingly almost as highly conserved as the coding regions. Both coding and 3'-noncoding portions appear to be under a similar degree of selective pressure and evolutionary constraint to remain constant.