Isolation and characterization of a complementary DNA clone for an algal pre-apoplastocyanin

We have isolated a cDNA clone for the Chlamydomonas reinhardtii pre-apoplastocyanin. The sequence contains codons for the complete pre-protein including a two-domain, lumen-targeting transit sequence and the mature apoprotein. The transit sequence (47 amino acids) is the shortest one described for chloroplast lumenal proteins, and like other C. reinhardtii lumen-targeting transit sequences appears to lack an uncharged amino-terminal domain usually present in plant lumen-directing sequences. The mature protein is deduced to be 98 amino acids in length and shows highest primary sequence similarity (74-76% identity) to other unicellular algal plastocyanins. Southern hybridization analysis of C. reinhardtii genomic DNA indicates the presence of a single nuclear gene, as is the case for all other plastocyanin genes characterized to date, although the algal gene might be interrupted. Codon usage in this gene reflects the high GC content of C. reinhardtii nuclear DNA, but is more highly biased than that found in the C. reinhardtii copper-repressible gene for the functionally equivalent pre-apocytochrome c552 (perhaps contributing to the more efficient synthesis in vivo of plastocyanin over cytochrome c552). The deduced physical properties of this plastocyanin are compared to those of the C. reinhardtii plastidic cytochrome c552.     

Isolation and characterization of oat globulin messenger RNA

When polyadenylated RNA, isolated from membrane-bound polysomes extracted from developing oat (Avena sativa L.) seeds, was translated in vitro in the rabbit reticulocyte system, two polypeptides of about 58 and 60 kilodaltons were immunoprecipitated by anti-oat globulin antibody. No electrophoretic bands corresponding to the 40 and 20 kilodalton polypeptides of oat globulin were present. However, when in vivo labeled extracts were immunoprecipitated with anti-oat globulin antibody, three groups of polypeptides (60, 40, and 20 kilodaltons) were present. It therefore seems probable that the two large polypeptides (58 and 60 kilodaltons) were precursors of the 40 and 20 kilodalton polypeptides. When the polyadenylated RNA coding for these polypeptides was size fractionated on a sucrose density gradient, it sedimented near the 18S region of the gradient. Translation of the RNA from the gradient fractions and immunoprecipitation of translation products indicated that the template for the 58 to 60 kilodalton `putative' precursors of oat globulin was probably the RNA which was approximately 18S in size.     

Isolation and characterization of collagen messenger RNA*.

Chick embryo collagen-synthesizing polysomes were isolated by differential centrifugation. RNA extracted from these particles was chromatographed in oligo(dT)-cellulose solumns and the mRNA thus obtained characterized as collagen mRNA by its electrophoetical mobility in acrylamide gels (equivalent to 1.05 x 10-6 daltons) and its effect upon a cell-free system derived from Krebs ascites tumor cells. The incorporation of 3H-proline was markedly dependent upon rabbit reticulocyte initiation factors and inhibited by initiation inhibitors such as aurintricaboxilate and pyrocatechol violet. The incorporation product was characterized as collagen by its lack of tryptophan, digestibility by purified bacterial collagenase, and by its co-chromatography with unlabled chick collagen in Sephadex G-200 and CM-cellulose columns.     

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.     

Isolation and partial characterization of poly (A)-containing 7.5S messenger RNA from rat liver mitochondria.

Poly(A)-containing low molecular weight (7.5S) messenger RNA was isolated in a highly purified form from both polyribosomes and post-polysomal supernatant of rat liver mitochondria. Both mRNA's contain rather short poly(A) tracts (40-70 mononucleotides) according to a profile of their elution from poly(U)-Sepharose column with a gradient of formamide concentration. Both mRNA's when added to a preincubated mitochondrial lysate programmed the synthesis of a hydrophobic polypeptide of a molecular weight about 9000 daltons which was soluble in the neutral chloroform-methanol mixture.     

Isolation and characterization of galanin from sheep brain.

Galanin is a well-known, naturally occurring peptide, which has been characterized from both cDNA sequences and direct peptide analysis. Previous structural studies have been made using intestinally derived material. This report concerns galanin isolation from sheep brain and its sequence determination. Sheep galanin shows great similarity to pig galanin, differing by one amino acid substitution, that being a histidine residue, as in cow and rat galanin, instead of tyrosine at position 26.     

Isolation of sheep anterior pituitary messenger RNA and its translation in a heterologous cell-free system

A preparation rich in the specific messenger RNA involved in the synthesis of prolactin from sheep anterior pituitary glands was obtained by employing both the immunochemical and affinity techniques. A dose-dependent and efficient stimulation of protein synthesis by the isolated total pituitary RNA as well as poly (A) rich RNA were achieved with the reticulocyte system. The synthesis of prolactin as one of the translational products of this cell-free system was established by specific immunoprecipitation followed by resolution on sodium dodecyl sulphate polyacrylamide gel electrophoresis.     

Alcohol dehydrogenase in Drosophila: isolation and characterization of messenger RNA and cDNA clone

The mRNA for alcohol dehydrogenase (ADH) in D. melanogaster has been identified by translation in a cell-free system. The in vitro synthesized polypeptide, specifically precipitated by anti-ADH antibody, has identical subunit molecular weight (25,000 daltons) and tryptic peptide profile to the in vivo synthesized ADH. The poly A containing ADH-mRNA has been purified by specific precipitation of ADH-polysomes using anti-ADH antibody and S. aureus. Transformation of E. coli with the dA-tailed ADH-mRNA-complementary DNA hybrid annealed to the dT-tailed pBR322 yielded one plasmid which has been identified as the ADH-cDNA clone. The identification involved hybridization selection of ADH-mRNA and in vitro translation, in situ hybridization to the Adh locus on salivary gland polytene chromosomes and DNA sequencing. This ADH-cDNA plasmid contains 349 bases of the C-terminal protein coding and 180 bases of the 3' untranslated region.