Isolation of two cDNA clones coding for larval hemolymph proteins of Galleria mellonella

Galleria mellonella a group of four larval hemolymph proteins (LHP) (74, 76, 81 and 82 kDa), which had been earlier shown to be storage proteins, exhibit a stage-specific synthetic pattern. The 82 kDa LHP is synthesized only in day-3 to day-5 last instar larvae, while the other three LHPs are synthesized both in the penultimate (six) and the last instar larvae. None of these LHPs are synthesized in day-0 last instar. With a view to isolate one or more cDNA clones corresponding to these LHPs a cDNA library was prepared in pBR322 starting with poly(A)⁺ RNA from day-5 last instar larval fat body. By differential screening of 714 clones with poly(A)⁺ RNA 39 day-5 larval stage-specific clones were isolated. Two of these clones, designated as 26–38 and 17–36, had 1200–1300 base pair cDNA inserts. Their cDNA inserts did cross hybridize to each other, exhibited different restriction endonuclease digestion patterns and hybridized in northern blots to transcrips of different sizes, thereby suggesting that they represent two separate genes. In addition, the genomic fragments that hybridized in southern blots to the two cDNAs differed in their size. On translation, mRNAs hybrid selected by 26–38 and 17–36 cDNAs produced 76 and 79 kDa polypeptides respectively. Both these genes are expressed in the fat body but not in the midgut, silk glands, Malpighian tubules or carcass. While 26–38 was expressed both in the sixth and seventh (last) instars, 17–36 was expressed only in the last instar. On the basis of tissue and developmental stage specificity of their expression and the sizes of their hybrid selected translation products, these clones are tentatively identified as two LHP-specific cDNA clones. The genes coding for these LHPs appear to be single copy genes.     

Isolation and characterization of cDNA clones for plant cyclins.

We have isolated and sequenced a carrot cDNA and two soybean cDNAs encoding mitotic cyclin homologs. The soybean clones were derived from nearly identical cognate genes. The carrot cyclin and soybean cyclins were slightly more similar to A-type and B-type cyclins thus far defined, respectively. However, they had divergent amino acid sequences in the portion that is most highly conserved in known cyclins and we could not easily include them in either of the phylogenetic types. Since the homology between carrot and soybean cyclins was low, each of them might define a novel and distinct type. The mRNA of carrot cyclin, 1.5 kb in length, was expressed concomitant with somatic embryogenesis of cultured cells. Expression of soybean cyclin mRNAs, 1.6 kb in length, was localized in proliferating parts of seedlings. As in the case of cyclin genes of marine invertebrates, microinjection of a synthetic mRNA for the soybean cyclin induced the maturation of Xenopus oocytes. Other cyclin genes may be present because, on Southern blot analysis of soybean genomic DNA, the isolated soybean cDNA probe hybridized with additional genes under low stringency.     

Isolation and characterization of cDNA clones for mouse Ly-9.

We describe the production and characterization of a mouse mAb, S-450-33.2, recognizing the Ly-9.2 specificity. This mAb was used to purify Ly-9 molecules from lymphoid cell lines, and the amino-terminal amino acids were determined. The mAb was also used in a eukaryotic expression system, to isolate cDNA clones encoding Ly-9. Analysis of RNA showed that Ly-9 expression is lymphocyte specific, as determined by the presence of a single hybridizing 2.4-kb species found only in lymphoid cells. Genomic DNA analysis indicated that Ly-9 is encoded by a single-copy gene of 10 to 15 kb. The predicted polypeptide belongs to the Ig superfamily of cell surface molecules with four extracellular Ig-like domains, i.e., a non-disulfide-bonded V domain, a truncated C2 domain with two disulfide bonds, a second non-disulfide-bonded V domain, and a truncated C2 domain with two disulfide bonds (V-C2-V-C2). The sequence data also support the view that Ly-9 belongs to the subgroup of the Ig superfamily that includes Bcm-1, CD2, and LFA-3.     

Up-regulation of two cDNA clones encoding metallothionein-like proteins in apple fruit during cool storage

We are investigating the molecular basis of low temperature responses in apples, by identifying and characterising fruit genes which show altered expression in response to cool-storage, Two independent cold-regulated clones (pAMTI and pAMT2) were isolated from a cDNA library derived from cool-stored apple ( Malus domestics Borkh. cv. Granny Smith) fruit. These clones share only 27% amino acid identity with each other, but both show high similarity to plant metallothionein (MT)-like proteins. The polypeptide encoded by pAMTI shares similarity with type 2 MT-like sequences, while that encoded by pAMT2 is similar to others which share a different distribution of cysteine residues. We suggest, these form a 'type 3' group of MT-like clones. Genomic Southern analysis confirmed that there is a family of MT-like genes in apple. There are differing patterns of pAMTI and pAMT2 expression during apple fruit development, amt 1 RNA was abundant in flowers and during the early stages of development, and decreased as the fruit approached maturity, while amt 2 RNA was barely detectable in flowers and young fruit and accumulated with fruit development. In ripe fruit. amt 1. expression was up-regulated, while amt 2 expression was down-regulated. In leaves, both genes showed increased expression with leaf age. In Granny Smith, Cox's Orange Pippin and Braeburn apple cultivars. both genes were up-regulated in cool-stored fruit. In Granny Smith contical tissue, amt RNA levels were elevated within the first 45 min at both 0.5°C and 4°C, but not at 12.5°C. The different patterns of amt 1 and amt 2 expression during fruit development and in different tissues suggest that the respective genes have distinct controlling elements and may be functionally different. The in vivo roles of the encoded polypeptides, particularly in relation to chilling tolerance or acclimation, are as yet unknown.     

Human alpha-L-fucosidase: complete coding sequence from cDNA clones

The human lysosomal storage disorder fucosidosis results from the deficiency of alpha-L-fucosidase, a lysosomal enzyme essential for the catabolism of oligosaccharides containing alpha-L-fucosides. cDNA clones coding for human alpha-L-fucosidase have been isolated from lambda gt10 and lambda gt11 cDNA libraries derived from human liver, placenta and colon. Compilation of cDNA sequences results in a nucleotide sequence of 2053 base pairs encoding alpha-L-fucosidase. The sequence contains an open reading frame of 461 amino acids beginning with the first in-frame methionine and includes 439 amino acids which comprise the mature protein in addition to a hydrophobic signal peptide sequence of 22 amino acids.     

Rabbit C-reactive protein. Biosynthesis and characterization of cDNA clones

To study the biosynthesis of rabbit C-reactive protein (CRP), a cDNA library was constructed from CRP mRNA-enriched polysomal poly(A) RNA. Four recombinant plasmids, designated pCX9, pCX23, pCX28, and pCX39, from 39 positive clones were sequenced and found to represent overlapping clones. DNA sequencing of CRP cDNA and primer extension of the 5'-end of CRP mRNA have demonstrated that the complete length of rabbit CRP mRNA consists of 2331 nucleotides and a terminal poly(A) segment. Analysis of the resulting sequence indicated that rabbit CRP mRNA contained a 5'-noncoding region of 107 nucleotides, a leader sequence encoding 20 amino acids, a coding region covering 205 amino acids, and a 3'-noncoding region of 1549 nucleotides. The 3'-noncoding region contained a consensus AAUAAA sequence that is 105 nucleotides upstream from the 3'-terminal poly(A) segment. Using an in vitro translation system, we have confirmed that CRP is synthesized as a precursor polypeptide (Mr approximately equal to 26,000) which undergoes processing to form the mature polypeptide (Mr approximately equal to 23,500). The CRP precursor failed to display a calcium-dependent affinity for phosphorylcholine ligand as demonstrated by mature CRP, suggesting that the phosphorylcholine-binding site of CRP only formed after processing. Northern blot analysis suggested that following induction with turpentine, liver was the only site where CRP mRNA synthesis could be demonstrated and that the change in mRNA concentration correlated with the course of CRP production. Southern blot analysis of liver genomic DNA indicated a single gene copy for CRP.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.     

Isolation and characterization of expressible cDNA clones for mouse Thy-1: a model system for cDNA expression of cell surface proteins

By using the mouse Thy-1 gene as a model, we have developed a procedure to distinguish functional vs nonfunctional cDNA of lymphocyte surface antigens by transfecting COS-7 monkey cells and testing for expression of cell surface products encoded by the cDNA inserts. By cross-hybridization with a mouse Thy-1 probe, we isolated cDNA clones from a pcD-expression library prepared from mRNA of C5 cells. Two functional clones were distinguished from the remainder by detection of Thy-1.2 on the surface of 0.5% of COS-7 cells transiently transfected by the DEAE-Dextran method. Inclusion of chloroquine in the transfection procedure greatly facilitated the detection of functional cDNA by raising the percentage of expressing cells to 30%. Nucleotide sequencing of one functional cDNA, about 1700 bp long, confirmed that the gene encodes a protein whose sequence agrees with the published Thy-1.2 protein sequence with the additional 31 amino acids attached at the COOH-terminus. A 75 bp 5' untranslated region preceding the coding region contains 50 bp not found in the genomic clones. Comparison indicates that one or more introns are present in the 5' untranslated region, but are not found in the mature mRNA. The first exon may be separated by at least 1 kb intron from the initiation codon. Because the expressible clones are approximately the size of the mRNA seen on Northern blots, we believe that these clones are nearly full-length cDNA. Dilution experiments indicate that this strategy should also be useful for identifying functional cDNA clones for cell surface proteins solely on the basis of their expression in mammalian cells.     

Isolation of cDNA clones coding for humoral lectin of silkworm, Bombyx mori, larvae.

In the hemolymph of the silkworm, Bombyx mori, lectin with hemagglutinating activity against sheep red blood cells increases at larval-larval ecdysis and at spinning stage (Suzuki and Natori, 1983) and is induced by infection with cytoplasmic polyhedrosis virus. A Bombyx lectin polypeptide with molecular weight approx 280K is responsible for hemagglutinating activity, since antiserum raised against this polypeptide inhibited hemagglutinating activity. The site of synthesis of Bombyx lectin was determined by primary tissue cultures of fat body and hemocytes. A hemagglutinating activity assay demonstrated that hemocyte is responsible for the release of hemagglutinin into the culture medium. Isolation of cDNA clones coding for Bombyx lectin was carried out on the cDNA library prepared in an expression vector lambda gt11 starting with poly(A)+ RNA from spinning larval hemocytes. As a result of immunoscreening, several positive clones were obtained, and the cDNA clones were characterized.