Isolation of cDNA clones for human adenosine deaminase

Clones encoding human adenosine deaminase (ADA) were isolated from a cDNA library made from the lymphoblastoid cell line MOLT-4. The isolation procedure was based on the selection of clones hybridizing with a radioactive probe complementary to an RNA preparation, which had been highly enriched in ADA-specific mRNA. The latter RNA preparation was obtained by size-fractionating MOLT-4 RNA and selecting fractions that were translatable into ADA. The assay for the presence of ADA in the in vitro translation products, was based on immunoprecipitation with a specific anti-ADA serum. The antiserum used was shown to precipitate a 42-kDal protein with the properties of ADA. Positive clones were further screened by means of hybrid-released in vitro translation assays. Two clones were obtained which were able to select mRNA that could be translated into a 42-kDal protein immunoprecipitable with the ADA-antiserum. By use of Southern blots containing DNA from somatic cell hybrids, one of these ADA cDNA clones was assigned to the human chromosome 20 known to contain the ADA gene.     

DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita

Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles. However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision. The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5′- and 3′-ends were localized by nuclease S1 mapping, leading to a size of 3277 nt. The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C. The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases. The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs. Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3′-part of the SSU rRNA. This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility. This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision.     

Construction of a cDNA clone containing the entire coding region of the human liver-type phosphofructokinase

Genomic fragments containing coding sequences of the human liver phosphofructokinase (PFKL) were used to screen cDNA libraries and several clones corresponding to PFKL were isolated. Three overlapping cDNA clones spanning the entire coding region were stitched together yielding the plasmid pG-cPFKL3.0 containing a 3Kb PFKL cDNA down stream of the T7 promoter. To assess its coding capacity pG-cPFKL3.0 was transcribed by T7 RNA polymerase and the RNA product was used to program an in vitro translation system. An 80KDa polypeptide which co-migrated with an in vivo labeled PFKL and was recognized by anti PFKL antibodies was synthesized. These results show that the cDNA clone pG-cPFKL3.0 contains the entire coding region of the human PFKL.     

Human adenosine deaminase. cDNA and complete primary amino acid sequence

A previously cloned partial adenosine deaminase cDNA insert (0.8 kilobase) was used to clone additional nucleotide sequences from human HPB ALL cDNA libraries. cDNA encompassing the entire coding, and 3'-untranslated regions as well as nearly all of the 5'-untranslated region was obtained. The complete amino acid sequence of the enzyme deduced from the cDNA sequence and protein sequencing consists of 362 amino acids, excluding the initiator Met, and accounts for Mr = 40,638. Secondary structure predictions assign adenosine deaminase to the alpha/beta class of proteins. Northern blot analysis with a cDNA probe showed adenosine deaminase mRNA to be present in normal to above normal amounts in B-lymphoblasts derived from adenosine deaminase-deficient patients with severe combined immunodeficiency disease. Knowledge of the cDNA and primary amino acid sequence of adenosine deaminase will be pivotal in further defining the genetic abnormality and its functional consequences in adenosine deaminase expression defects.     

A point mutation in the 5′ splice region of intron 7 causes a deletion of exon 7 in adenosine deaminase mRNA

An adenosine deaminase (ADA;EC 3.5.4.4)-deficient B lymphoblastoid cell line BAD05 derived from a Japanese patient with severe combined immunodeficiency was characterized. As previously reported, one allele of BAD05 expresses undetectable ADA mRNA, and the other allele produces an aberrant mRNA without exon 7. Genomic ADA DNA of BAD05 spanning from a portion of exon 6 to a portion of exon 8 was amplified by PCR. The amplified fragments were cloned into a vector, and 8 clones were isolated and sequenced. The analytical result showed a single base change of G to A at the invariant 5′ GT of intron 7 of ADA gene in one allele of BAD05, which accounts for the elimination of exon 7 during splicing. © 1993 Wiley-Liss, Inc.     

Sequence analysis of a cloned cDNA coding for bovine seminal ribonuclease

The sequence of a cloned cDNA coding for bovine seminal ribonuclease, an enzyme secreted in the bull seminal vesicles, was determined. The cDNA starts at the amino acid residue 47 and terminates 12 nucleotides beyond the consensus sequence AAUAAA in the 3' non-coding region of the mRNA. Northern blotting analysis shows that the mRNA for bovine seminal ribonuclease consists of about 950 nucleotides, a value that is similar to that of other mRNAs coding for ribonucleases of the pancreatic type.