Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences.

A cDNA library was derived from the poly(A)(+) RNA of young tomato leaves. The library was cloned in a λgt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5' end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)(+) RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.

An experimental approach to enumerate the genes coding for immunoglobulin variable-regions.

Critical to our understanding of the immune system diversity is the determination of the number of germ line V genes. The total number of V genes is given by the product: number of subgroups x number of germ line genes per subgroup. Studies of kappa chains and of embryonic DNA indicate 5-10 V genes per subgroup. Statistical analysis of the limited sequence data of mouse kappa chains suggest about 50 V kappa subgroups. We report here a general approach for direct estimation of the number of VL and VH subgroups expressed in normal spleen, and present data for V kappa. The kappa mRNA of the spleen is a heterogeneous population where different V kappa are linked to the same C kappa, i.e. C kappa equals total V kappa. The ratio C kappa/distinct V kappa approximates the number of subgroups since V kappa of the same subgroup cross hybridize while V kappa of different subgroups do not. This ratio was determined by molecular hybridization of cloned C kappa and V kappa DNA probes with spleen mRNA. The results indicate the expression of 280 V kappa subgroups in mouse. Assuming an average of 7 genes per subgroup, we estimate about 2000 V kappa germ line genes.

Synthesis of part of a mouse immunoglobulin light chain in a bacterial clone.

We have cloned double stranded cDNA sequences encoding a mouse immunoglobulin light chain (L-321) into the PstI site of the beta-lactamase gene of plasmid pBR322 by the oligo (dG)-oligo (dC) tailing procedure. Escherichia coli X1776 transformed by the recombinant plasmids were screened for the expression of L-321 antigenic determinants by a newly developed in situ radio-immunoassay. One out of seven transformants screened was found to synthesize an L-chain like protein. Each bacterial cell produces about 550 molecules of the L-chain sequence. Preferential segregation of the L-chain sequence to the periplasmic space suggest covalent attachment of the L-chain sequence to the N-terminal portion of beta-lactamase. Restriction mapping of the plasmid DNA isolated from the positive clone indicated the presence of a DNA sequence coding for the entire constant region and extending into the variable region for a length corresponding to about 40 amino acid residues. The orientation of the cloned cDNA with respect to the plasmid DNA is compatible with the formation of a fused beta-lactamase-L-321 peptide.

Cell-free synthesis of rat parotid preamylase.

Poly(A)-containing RNA from rat parotid gland directs the cell-free synthesis of several products in the reticulocyte lysate translation system including a very prominent 58,000-dalton polypeptide which is immunoreactive with anti-alpha-amylase. Purified alpha-amylase has a molecular weight estimated as 56,000 daltons. The 58,000-dalton, cell-free product and alpha-amylase share common peptides as determined by analysis of their limited proteolysis digests. The cross-reactivity and peptide homology suggest that the cell-free product may be a precursor of mature alpha-amylase. While the NH2 terminus of alpha-amylase is blocked, that of the 58,000-dalton product evidently is not, and automated sequence analysis has yielded its partial sequence as: Met-X-Phe-Phe-Leu-Leu-Leu-X-Leu-Ile-X-Leu-X-X-X-X-X-X-X-X-X-Phe-X-X-X-X-X-Ile-X-X-Leu-Phe. The highly hydrophobic nature of the NH2 terminus of the 58,000-dalton, cell-free product suggests that, like other secreted polypeptides, the extra piece may play a role in the transport and secretion of the mature alpha-amylase.