Hybrid-selected Translation Research Articles

Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor-Releasing Factor, β-1,3-Endoglucanase, in Soybean

Soybean (Glycine max) beta-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced beta-1,3-endoglucanase activity to 2- to 3-fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean beta-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable beta-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a lambda-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with beta-1,3-endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of beta-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean beta-1,3-endoglucanase exhibited 53% amino acid homology to a beta-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a beta-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable beta-1,3-endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription.

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B′) and isolation of its cDNA and genomic DNA

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.

Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum

Leaves of the resurrection plant Craterostigma plantagineum Hochst, can be desiccated up to 1% relative water content and are still viable after rehydration. To clone genes related to this extreme desiccation tolerance, an in-vitro system was first developed which allows the induction of the same resurrection response in callus tissue upon treatment with abscisic acid (ABA). Several proteins and in-vitro-synthesized polypeptides were then identified which can be induced both in desiccation-tolerant, naturally dried leaves and in ABA-treated calli surviving after rehydration. Complementary-DNA clones corresponding to mRNAs expressed only in desiccation-tolerant tissues were obtained and classified into several gene families. In hybrid-selected translation experiments, representative cDNA clones were associated with water stress and ABA-inducible polypeptides abundantly expressed in dried leaves and ABA-treated calli. The expression pattern of several of these abundant transcripts was analyzed in RNA-hybridization experiments. Upon stress or ABA treatment the transcription levels increased rapidly, but they declined after relief from the stress state. This, together with data on genomic copy numbers indicated that a set of abundantly expressed genes are involved in the desiccation process of resurrection plants. Data on endogenous ABA contents before and after stress applications and on the physiological effects of exogenous ABA treatments indicate that in Craterostigma plantagineum the induction of an extreme desiccation tolerance is mediated by this plant hormone.

Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single mRNA with two alternative in-phase aug initiation sites

By use of anticytosolic fumarase antibody, a cDNA clone was isolated from a rat liver cDNA library in the expression vector λgt11 and in the pBR 322 vector. A clone with an insert of about 1.7 kbp was isolated. Nucleotide sequence analysis of the insert revealed that the cDNA contained a noncoding region composed of 25 nucleotides in the 5′ terminus, the coding region composed of 1,521 nucleotides, and the 3′ nontranslated region composed of 43 nucleotides followed by a poly(A) + tail. The open reading frame encoded a polypeptide of 507 amino acid residues (predicted M r = 54,462), which contained an additional sequence composed of 41 amino acid residues on the N-terminus of the mitochondrial mature fumarase (the presequence). Thus, this reading frame was concluded to encode the precursor of mitochondrial fumarase. The amino acid sequence predicted from the nucleotide sequence contained all the amino acid sequences of 12 proteolytic polypeptides obtained by digestion of purified mitochondrial fumarase with V8 protease. The total amino acid sequence of the mitochondrial fumarase also contained all the sequences of 14 proteolytic peptides prepared from the cytosolic fumarase, indicating that the amino acid sequences of these two isozymes are identical. Furthermore, the results obtained by hybrid-selected translation, Northern blot and primer-extension analyses using appropriate cDNA segments prepared from fumarase cDNA (1.7 kbp) as the probe or primer suggested a possibility that both precursors of the mitochondrial and cytosolic fumarases were synthesized with one species of mRNA having base sequence coding presequence of the mitochondrial fumarase by unknown post-transcriptional mechanism(s). Rat liver cells may contain a specific RNA(18S) modulating the translational activity of mRNA for fumarase. This RNA(s), which was contained in poly(A) − fraction, was partially purified by high-performance gel filtration. The partially purified RNA(s) suppressed the translational activity of the cytosolic fumarase, whereas the translational activity of the mitochondrial one was accelerated by this RNA(s).

Expression of genes encoding oleosin isoforms in the embryos of maturing maize kernels

Oleosins are small hydrophobic proteins localized on the surface of oil bodies in diverse seeds. A cDNA clone of maize ( Zea mays L.) oleosin KD 18 was obtained by screening a cDNA library of maturing maize embryos with cDNA probes obtained by reverse transcription of size-fractionated poly(A) +RNA, and identifying the KD 18 clones using hybrid-select translation followed by immunorecognition of the translation products. Northern hybridization analyses using KD 18 cDNA as a probe were employed to study the levels of KD 18 mRNA. Two species of KD 18 mRNA were found; the major one had 1.1 kb and the minor one contained 1.0 kb. The presence of KD 18 mRNA was tissue specific and was found only in the maturing embryo. The level of KD 18 mRNA in the embryo increased during seed maturation and reached a peak 40 days after pollination. It declined rapidly as the kernel matured and was not detectable 2 days after seed imbibition. Abscisic acid applied to imbibing mature kernels inhibited germination and, at the same time, enhanced the level of KD 18 mRNA and prolonged its presence. An examination of the published genomic sequence of the KD 18 gene revealed putative ABA-responsive sequences in the 5′ upstream region of the coding sequence. Southern hybridization of restriction enzyme digests of genomic DNA showed two possible KD 18 genes. No specific amplification of the KD 18 gene was apparent in the maturing embryo. The pattern of gene expression is similar, although not identical, to that of oleosin KD 16 gene in the same maize embryo. The coordinate expression of the two maize oleosin genes emphasizes the close similarity among oleosin isoforms.

Apparent expression of small single copy cpDNA region in senescent chloroplasts

Tobacco dpDNA fragments have been used as probes for hybrid select translation in a protein synthesis system (derived from nuclease-treated senescent barley chloroplasts) depending on externally added mRNA retaining translational specificity (as it preferently translated mRNAs for polypeptides synthesized during senescence). Selections from crude mRNAs (isolated from senescent chloroplasts containing some mRNAs not translated in vivo) and translations indicated that most genes for the polypeptides synthesized early in leaf senescence map in the SSC cpDNA region. This region is poorly expressed during chloroplast biogenesis and includes four genes ( ndh) homologous to those for components of mitochondrial NADH dehydrogenase. Accordingly, senescent chloroplasts show high ferricyanide reducing activity, probably related to NADH dehydrogenase. Apparently, chloroplast ndh gene expression is activated during leaf senescence at the level of translation.

Recombinant baculoviruses as vectors for identifying proteins encoded by intron-containing members of complex multigene families.

Using a transfer vector derived from Bombyx mori nuclear polyhedrosis virus (BmNPV), we have constructed recombinant baculoviruses that contain complete silk moth chorion chromosomal genes encoding high-cysteine proteins under the control of the polyhedrin promoter. Silk moth tissue culture cells infected with these recombinant viruses were found to contain abundant RNA sequences of sizes similar to those of the authentic chorion mRNAs. Chorion transcripts present in infected cells were initiated almost exclusively at the cap site of the polyhedrin start site. Primer extension and RNase protection experiments revealed that a considerable proportion of the resultant transcripts were spliced at the same sites as those utilized in follicular cells for the production of functional chorion mRNA. Electrophoretic analysis and immunoprecipitation of the proteins of host cells infected with the recombinant viruses revealed the presence of the corresponding chorion proteins. We conclude that baculovirus vectors can be used for expressing efficiently not only cDNAs or simple genes devoid of intervening sequences but also intron-containing chromosomal genes. Thus, recombinant baculoviruses offer a powerful alternative to hybrid-selected translation, particularly when the identification of proteins encoded by members of complex multigene families is required.

Characterization of cDNA clones of host RNAs isolated from cauliflower mosaic virus-infected turnip leaves

We have characterized changes in host RNA populations during infection of turnip plants by cauliflower mosaic virus (CaMV) using a molecular cloning technique. A lambda gt10 library of complementary DNA (cDNA) clones was generated corresponding to host RNAs isolated from systemically-infected leaves during the vein-clearing stage. Specific clones representing RNAs with altered levels were selected from the library by differential screening with cloned hybridization probes derived from non-infected and infected plants. Three clones were characterized in detail. Using cDNA to probe RNA species by Northern hybridization, clones pM25 and pM4 detected RNA molecules each of 850 bases whose level decreased and increased respectively during infection. Hybrid select translation demonstrated that these RNAs encoded polypeptides of 22 K and 18 K molecular weights. Following DNA sequencing and comparison with a computer database, clone pM25 was identified as the ribulose 1,5-bisphosphate carboxylase small subunit polypeptide whilst no match was found for pM4. A further clone (pM53) hybridized with a complex population of RNAs that were identified as chloroplast ribosomal RNA species.

A major transcript in the response of Neurospora crassa to protein synthesis inhibition by cycloheximide

In Neurospora crassa grown in minimal medium with 2 μ M cycloheximide, the expression of several genes was shown to be turned on. We have isolated one of those genes coding for a major transcript in this response, which newly appears after addition of cycloheximide and remains present at a high level for a long period. An open coding region for a 70-kDa protein was demonstrated by hybrid-selected translation, in agreement with the in vivo labeling of a protein with the same mobility in cycloheximide-induced cultures. Even 48 h after cycloheximide addition, CI-1-specific mRNA was bound to polysomal fractions. Our results also suggest that DNA sequences analogous to N. crassa CI-1 gene are present in other species. In N. crassa, CI-1 gene would not be induced by heat shock or starvation.

Expression of different members of heat shock protein 70 gene family in liver and hepatomas.

The levels of expression of some genes of the HSP 70 family have been assessed in rat liver and in a series of transplantable hepatomas with different growth rates, subjected to heat shock in vivo. For this purpose, the mRNAs for the constitutive cognate HSC 73, the heat-inducible HSP 70 and the glucose-regulated GRP 78 have been analyzed by: (i) translation in reticulocyte lysates; (ii) hybrid-selected translation, and (iii) Northern blot analysis. In comparison with the liver, the fast-growing 3924A hepatoma has an increased constitutive amount of HSC 73 mRNA and a lower induction of HSP 70 mRNA after heat shock. The behavior of the 9618A slow-growing hepatoma is more similar to that of the liver, indicating that the changes detected in the fast-growing hepatoma are correlated to the high growth rate of the tumor rather than to carcinogenesis. This conclusion is reinforced by the results obtained with Yoshida AH-130 cells, growing at two different rates imposed by the environment in which they develop. When the Yoshida hepatoma grows rapidly in the peritoneal cavity, constitutive expression of HSC 73 mRNA is high and the inducibility of HSP 70 mRNA is poor: the opposite occurs when the tumor grows slowly in the subcutaneous compartment. The amount of GRP 78 mRNA increases progressively from the liver to the fast-growing hepatoma. The level of HSC 73 mRNA seems to correlate with the methylation state of the corresponding gene.

Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene

By use of anti-cytosolic fumarase antibody, a cDNA clone was isolated from a rat liver cDNA library in the expression vector lambda gt11 and in the pBR 322 vector. A clone with an insert of about 1.7 kilobases was isolated. Nucleotide sequence analysis of the insert revealed that the cDNA contained a 5'-noncoding region of 25 nucleotides, the coding region of 1,521 nucleotides, and a 3'-nontranslated region of 43 nucleotides followed by a poly(A)+ tail. The open reading frame encoded a polypeptide of 507-amino acid residues (predicted Mr = 54,462), which contained an additional sequence of 41-amino acid residues on the NH2 terminus of the mitochondrial mature fumarase (the presequence). Thus, this reading frame was concluded to encode the precursor of mitochondrial fumarase. The amino acid sequence predicted from the nucleotide sequence contained all the amino acid sequences of 12 proteolytic polypeptides produced by digestion of purified mitochondrial fumarase with V8 protease. The total amino acid sequence of the mitochondrial fumarase also contained all the sequences of 14 proteolytic peptides obtained from the cytosolic fumarase, indicating that the amino acid sequences of these two isozymes are identical. Furthermore, the results obtained by hybrid-selected translation, Northern and Southern blot, and primer-extension analyses using appropriate cDNA segments prepared with fumarase cDNA (1.7 kilobases) as a probe or primer suggested that the mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene and a possibility that the precursors of both these fumarases were synthesized by one species of mRNA having a base sequence coding the presequence of the mitochondrial fumarase by some unknown post-transcriptional mechanism(s).

Utilization of Purified Pertinent Fungal Enzymes for Development of Probes to Identify Genes Responsible for Aflatoxin Biosynthesis

AbstractThe observation that O-methylsterigmatocystin (OMST) is a metabolite in the aflatoxin biosynthetic pathway between sterigmatocystin (ST) and aflatoxin B1 (AFB1) led to the discovery of two enzyme activities responsible for the late stages of aflatoxin synthesis: 1) a methyltransferase (MT) catalyzing the conversion of ST to OMST, and 2) an oxidoreductase (OR) which converts OMST to AFB1. The anionic MT (168 KDa; two subunits, 110 KDa and 58 KDa) has been purified to homogeneity and well characterized. The oxidoreductase has been partially purified. Polyclonal antibodies against MT have been prepared and amino acid composition of MT determined. A cDNA library, constructed from mRNA isolated from Aspergillus parasiticus mycelia during the onset of AFB1 biosynthesis, is screened by a standard Hybrid Select Translation procedure to identify the genes responsible for the biosynthetic steps at the terminus of the aflatoxin pathway.

Isolation and sequence determination of a cDNA clone for rat peroxisomal urate oxidase: liver-specific expression in the rat.

Urate oxidase (UOxase; urate:oxygen oxidoreductase, EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but is absent in humans and certain primates. A cDNA clone for UOxase containing an insert of 1.3 kilobases (kb) was isolated from a lambda gt11 cDNA library prepared from rat liver mRNA. This recombinant clone with a 1283-nucleotide insert has sequence for 97% of the coding region together with 401 nucleotides of the 3'-untranslated region of the mRNA. The identity of UOxase cDNA clone was verified by analyzing the fusion protein, immunocytochemical localization with epitope-selected antibody, and hybrid-select translation analysis and by comparing sequences of four CNBr-cleaved peptides of the protein. Blot analysis revealed that the probe hybridizes to a single 1.5-kb mRNA species in the rat liver and a transplantable hepatocellular carcinoma. No UOxase mRNA was detected in 11 nonhepatic tissues of rat, suggesting tissue specificity of expression of this UOxase gene. Blot analysis of RNA from livers of rats treated with a peroxisome proliferator showed 2- to 3-fold increase in UOxase mRNA content, whereas the fatty acyl-CoA oxidase mRNA increased over 30-fold. Southern blot analysis of restriction enzyme digests of rat DNA suggests that there is a single copy of UOxase gene. Analysis of human genomic DNA revealed restriction fragments that are homologous to rat UOxase cDNA, although no UOxase mRNA was detected in human liver.

Cloning and Characterization of a Mouse Type I Hair Keratin cDNA

A cDNA library was prepared from poly(A)-containing C57BL/6J mouse hair-root-enriched mRNA. The library was screened using a radiolabeled nucleic acid probe prepared from a sheep wool, Type I keratin cDNA clone (SWK2). Clone MHKA-1 was shown to contain a mouse hair, Type I keratin cDNA insert by positive hybridization-selection translation assay, and by the corresponding deduced amino acid sequence. The size of the cDNA insert is 1585 bp (excluding homopolymer tails) and on the basis of Northern blot analysis it corresponds to a mRNA of approximately 1.6 Kb. The cloned cDNA sequence includes the entire coding region for a protein of 416 amino acids (including the initiation methionine). Comparison of the deduced amino acid sequence with that of sheep wool, Type I keratin (8c1) reveals an overall corresponding sequence identity of 87%. In contrast, the MHKA-1 protein is significantly less similar to non-hair Type I keratins. An additional 3 amino acids (adjacent proline residues) not present in the wool protein have been identified near the middle of the carboxy-terminal end of the mouse protein. MHKA-1 is the first of a series of mouse hair follicle cDNA clones to be identified and characterized that will enable us to study follicular regulatory mechanisms and the interrelationships among the proteins in the mammalian hair follicle.

A third prolactin-like protein expressed by the developing rat placenta: complementary deoxyribonucleic acid sequence and partial structure of the gene.

We have previously reported on the isolation and characterization of two PRL-related cDNA clones, rat placental lactogen II and rat PRL-like protein A which are among the more abundant mRNAs expressed int he late term rat placenta. In this paper we report the isolation and characterization of cDNA clones of a third abundant placental protein, the predicted amino acid sequence of which shows a 44% homology to rPRL. This protein which we have called rPRL-like protein B (rPLP-B) is different from, but related to, the other PRL related proteins which have been identified in the rat, mouse, and bovine placentas. Two rPLP-B mRNA transcripts of 0.9 and 1.2 kilobases are strongly expressed in essentially equal amounts from day 14 of pregnancy until term. Nucleotide sequence and hybrid select translation data predict one secreted, potentially glycosylated protein of approximately mol wt 27,000. Hybridization and primer extension studies show that the two transcripts differ in their 5'-untranslated regions. One of the cDNA clones isolated represents a portion of the unprocessed rPLP-B mRNA. All intron/exon boundaries in this clone are of the same splice class and occur in identical locations within the coding region as in the rPRL gene.

Isolation and characterization of a cDNA clone encoding rat 5-lipoxygenase.

A full-length cDNA clone encoding 5-lipoxygenase, a key enzyme in the formation of leukotrienes, was isolated from a rat basophilic leukemia cell lambda gt11 cDNA library. The 2.5-kilobase (kb) cDNA insert, whose identity was confirmed by hybrid-select translation and DNA sequence analysis, has a 2.0-kb open reading frame encoding a protein of Mr approximately 77,600 and includes 60 base pairs of 5'-untranslated region and 0.4 kb of 3'-untranslated region to the polyadenylation signal. The deduced amino acid sequence shows significant homology with published sequences for the rabbit reticulocyte lipoxygenase and soybean lipoxygenase-1; it also contains sequences similar to a consensus sequence found in several calcium-dependent membrane-binding proteins. The cDNA recognizes a 2.6-kb mRNA species which is detected in all tissues but is particularly abundant in RNA from lung.

CDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a cDNA encoding the peroxisomal trifunctional β-oxidation enzyme hydratase-dehydrogenase-epimerase (HDE) from the yeast Candida tropicalis pK233. Poly(A) + RNA isolated from C. tropicalis cells grown in oleic acid medium was used to construct a cDNA library in λgt11. The library was screened with a polyclonal antiserum against HDE. A recombinant was confirmed to encode HDE by hybridization-selection translation and immunoprecipitation. The HDE cDNA ( HDE) has a single open reading frame of 2718 nt, encoding a protein of 905 amino acids, not including the initiator methionine. The M r of the protein is 99 350. A partial gene duplication is believed to have occurred in the evolution of the HDE gene. Codon utilization in the gene is not random, with 86.0% of the amino acids specified by 23 preferentially used codons, a situation similar to that found in genes encoding peroxisomal catalase and the various fatty acyl-CoA oxidases from C. tropicalis. The increase in HDE activity in C. tropicalis cells grown in oleic acid medium as opposed to glucose medium is due, at least in part, to increased HDE-specific mRNA levels.

Fetal and variant .alpha.-fetoproteins are encoded by mRNAs that differ in sequence at the 5' end

The temperature-sensitive RLA209-15 fetal rat hepatocyte line grown at the nonpermissive temperature (40 degrees C, normal phenotype) produces authentic rat alpha-fetoproteins (AFPs) of 69K and 73K (fetal AFPs) which are encoded by a 2.2-kb mRNA. These cells also produce low levels of a 1.7-kb AFP mRNA and a 65K variant AFP when grown at the permissive temperature (33 degrees C, transformed phenotype). Hybrid-selected translation demonstrates that the 1.7-kb AFP mRNA encodes the 65K variant AFP. Northern blot hybridization and S1 nuclease analyses indicate that the 1.7-kb mRNA lacks sequences present in the first seven 5' exons of the 2.2-kb AFP mRNA. However, the 1.7- and 2.2-kb AFP mRNAs share common sequences extending from the beginning of the eighth exon (corresponding to nucleotide 873 of the fetal AFP mRNA) to the 3' end. Primer extension analysis suggests that the 1.7-kb RNA contains additional sequences 5' to the common regions shared by both AFP mRNAs. We have previously shown that adult rat liver produces a 1.7-kb AFP mRNA; we now report the isolation of a cDNA (ARFP5) encoding this variant AFP mRNA from an adult rat liver cDNA library. Restriction endonuclease mapping and sequence analysis of ARFP5 confirm that the 1.7- and 2.2-kb AFP mRNAs share similar sequences at the 3' region (approximately 1.1 kb). However, ARFP5 contains an additional 90 bp variant AFP mRNA-specific 5' sequence which is located in the seventh intron of the rat AFP gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cDNA sequences for LHCI apoproteins in Euglena gracilis: the mRNA encodes a large precursor containing several consecutive divergent polypeptides.

Screening of a lambda gt11 cDNA expression library with antibodies directed against LHCII allowed the detection of several clones which differ markedly from the previously described cDNAs encoding LHCII apoproteins. A Northern analysis revealed a transcript size of 4.2 kb, whereas clones encoding LHCII hybridize with mRNAs in the range of 7.5 kb. Nucleotide sequencing of 2 clones showed open reading frames of 530 and 331 codons, respectively. Within these reading frames, 5 analogous motifs can be delimited corresponding to coding regions for around 180 amino acids (molecular weight, 18-19 kDa). The 5 segments share between 50% and 80% homology. Comparison with a tomato LHCI sequence indicates conserved regions at the two ends, and a central part highly divergent and containing a large deletion. By hybrid-selected translation, followed by immunoprecipitation with a monoclonal antibody directed against LHCI apoprotein, a protein of around 100 kDa is obtained. In vivo, the same antibody recognizes peptides of around 20 kDa. These results, coupled with our previous observations concerning LHCII, confirm that in Euglena at least, some chloroplast proteins encoded in the nucleus are synthesized by large mRNAs containing the information for several consecutive divergent peptides. Implications for processing and chloroplast import are discussed.

Molecular cloning of a bovine immunoglobulin lambda chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) λ light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig λ chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig λ chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig A chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig λ chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5′ end of the bovine Ig λ chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Igλ mRNA of clone 1-14E is transcribed from the V λ pseudogene.