Molecular cloning of cDNA for Avena phytochrome

We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated Avena poly(A)⁺ RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A)⁺ RNA that had been either enriched in or depleted of phytochrome mRNA. Of the colonies hybridizing preferentially with the enriched probe, several contained plasmids that specifically selected phytochrome mRNA when assayed by hybridization-selection and translation. The largest such plasmid, pAP-2, was used to isolate clones from an Avena genomic library. One of these genomic clones was then used to screen a second cDNA library in an attempt to identify full-length phytochrome clones. The largest of the plasmids thus obtained, pAP-3, contains a 3.4-kilobasepair (kbp) insert, verified to contain phytochrome sequences by hybridization-selection and translation. Sequence analysis of pAP-2 and pAP-3 revealed that the two clones are identical in sequence through a 2.4-kbp region in which they overlap. However, the pAP-2 insert contains, in addition, 1.5 kbp of sequence of unknown origin, the apparent result of a recombination event. Blots of poly(A)⁺ RNA hybridized with ³²P-labeled pAP-2 or pAP-3 show a single mRNA band at 4.2 kilobases. Blot analysis of RNA from dark-grown and from red-irradiated tissue demonstrates that a previously reported light-induced decrease in translatable phytochrome mRNA results from a decrease in physical abundance of this mRNA.     

Cloning and structure determination of cDNA for cutinase, an enzyme involved in fungal penetration of plants

The primary structure of cutinase, an extracellular fungal enzyme involved in the penetration of plants by pathogenic fungi, has been determined from the nucleotide sequence of cloned cDNA. Clones containing cDNA made from poly(A)⁺ RNA isolated from fungal cultures induced to synthesize cutinase were screened for their ability to hybridize with the [³²P]cDNA for mRNA unique to the induced culture. The 75 cDNA clones thus identified were screened for the cutinase genetic code by hybrid-selected translation and examination of products with anti-cutinase IgG. This method yielded 15 clones containing cDNA for cutinase, and Southern blots showed that the size of the cDNA inserts ranged from 279 to 950 nucleotides. Blot analysis showed that cutinase mRNA contained 1050 nucleotides, indicating that the clone containing 950 nucleotides represented nearly the entire mRNA. This near-full-length cDNA and the restriction fragments subcloned from it were sequenced by a combination of the Maxam-Gilbert and the phage M13-dideoxy techniques. cDNAs from two other clones, containing the bulk of the coding region for cutinase, were also completely sequenced, and the results confirmed the sequence obtained with the first clone. A peptide isolated from a trypsin digest of cutinase was sequenced and the amino acid sequence as well as the initiation and termination codons were used to identify the coding region of the cDNA. The primary structure of the enzyme so far determined by amino acid sequencing (≈40% of the total) agreed completely with the nucleotide sequencing results. Thus, the complete primary structure of the mature enzyme and that of the signal peptide region were ascertained.     

Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening

The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-α-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)⁺ RNA isolated from ripe tomato fruit. The cDNA library was inserted into a λ-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.     

F1 and F2: Two similar genes regulated differently during development of Drosophila melanogaster

Screening a cDNA library of 2- to 3-day-old flies with poly(A)⁺ RNA from male and female flies, we were able to isolate a small number of clones hybridizing preferentially with RNA from female flies. Four cDNA clones, derived from a single mRNA species, proved to be highly abundant in female flies. When we screened a genomic library with the longest cDNA, we obtained two genomic clones, F1 and F2; F1 was a direct copy of the cDNA and F2 was obtained by cross-hybridization. A detailed analysis of these genomic clones revealed two independent genes coding for proteins of 50 kDa that are >90% homologous. RNA analysis with gene-specific probes from the 3′ untranslated region showed an expression of F1 in all stages of development with a 5- to 10-fold overexpression of this RNA in female flies compared with males. In contrast to F1, F2 is mainly expressed in late pupae and is expressed only at low levels in adult flies.     

Isolation of cloned cDNAs to auxin-responsive poly(A)RNAs of elongating soybean hypocotyl

Auxin-responsive cDNA clones have been isolated from a cDNA library prepared from elongating soybean hypocotyl poly(A)⁺RNA. The expression of two such sequences has been assessed by RNA blot hybridization analyses during normal developmental transitions in the soybean hypocotyl and during incubation of sections excised from the region of cell elongation. The concentrations of these poly(A)⁺RNAs are higher in the elongating zone than in the apical and mature zones of the hypocotyl. Both poly(A)⁺RNAs are depleted during incubation of the sections in the absence of auxin. The loss of one of these sequences (pJCW1) is prevented by the addition of auxin to the incubation medium while the other sequence (pJCW2) increases above the initial level in the presence of auxin. The addition of auxin to auxin-depleted tissue in which the sequences are depleted results in rapid accumulation of these poly(A)⁺RNAs; pJCW1 accumulates to the control level while pJCW2 increases well above the control level. These data along with others [Baulcombe, D. C. & Key, J. L. (1980) J. Biol. Chem. 255, 8907-8913] demonstrate directly a highly selective effect of auxin on the expression of a small number of mRNAs in tissues undergoing both cell elongation and cell division in response to auxin. Although the data are suggestive of a close association betwen auxin action and altered gene expression, a causal relationship is not established. It seems highly unlikely, however, that such specific effects of auxin on gene expression are unimportant in auxin physiology.     

Cloning and expression of cDNA for salmon growth hormone in Escherichia coli

cDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)⁺ RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in Escherichia coli carrying a plasmid in which the sGH cDNA was under control of the E. coli trp promoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from E. coli stimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.     

Isolation and sequence characterization of a cDNA clone of human antithrombin III.

A human liver cDNA library was constructed by using poly(A)-containing RNA isolated from a human liver biopsy specimen. This library is comprised of 40,000 independent transformants with an average inserted DNA length of 1,200 base pairs. By using the previously cloned baboon antithrombin III cDNA as a specific hybridization probe, greater than 30 human antithrombin III cDNA clones were identified from this library. The clone with the longest DNA insert was selected for sequence analysis. This antithrombin III cDNA clone contains 1,479 base pairs of inserted human DNA and was designated phATIII 113. It contains DNA sequences that code for a signal peptide and the entire mature antithrombin III protein which is comprised of 432 amino acid residues.     

Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine.

We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine. The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks. Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two [32P]cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks. We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA. With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA. The differential colony hybridization procedure using an enriched vs. a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.     

Localization of a PlA1 epitope to the amino terminal 66 residues of platelet glycoprotein IIIa.

A platelet glycoprotein (GP) IIIa epitope library was constructed by insertion of randomly cleaved GPIIIa cDNA fragments in the prokaryotic expression vector lambda gt22 and screened with purified anti-PlA1 antibodies for clones expressing a PlA1 epitope. Five independent clones were isolated and characterized by nucleotide sequencing. The smallest anti-PlA1 reactive clone obtained encoded the amino terminal 66 residues of mature GPIIIa. Substitution of leucine33 (PlA1) with a proline33 (PlA2) by in vitro mutagenesis resulted in the loss of anti-PlA1 reactivity; however, this clone still reacted with anti-GPIIIa polyclonal antibodies. These data indicate that a PlA1 alloantigenic epitope is located within a small, unglycosylated fragment of GPIIIa containing the polymorphism responsible for the PIA phenotype. Furthermore, these results prove that small recombinant mimics of a PlA1 epitope may be synthesized and used for detection of these alloantibodies.     

Androgen regulation of canine prostatic arginine esterase mRNA using cloned cDNA

Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.     

Cloned DNA sequences complementary to mRNAs encoding precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and a chlorophyll a/b binding polypeptide

Double-stranded cDNA was synthesized from pea poly(A)-containing mRNA and inserted into the Pst I site of the bacterial plasmid pBR322 by the addition of synthetic oligonucleotide linkers. Bacterial colonies containing recombinant plasmids were detected by hybridization to partially purified mRNAs and further characterized by cell-free translation of hybridization-selected mRNAs. To confirm the identity of cDNA clones encoding chloroplast polypeptides, we incubated translation products derived from complementary mRNAs with intact chloroplasts in vitro. After uptake, precursor polypeptides were converted to their mature size and identified by fractionation of the chloroplast stroma and thylakoid membranes. By using these procedures, we have isolated and characterized cDNA clones encoding the two major cytoplasmically synthesized chloroplast proteins: the small subunit of ribulose-1,5-bisphosphate carboxylase and a constituent polypeptide (polypeptide 15) of the light-harvesting chlorophyll a/b-protein complex. Similarly, a third cDNA clone was isolated and shown to encode a 22,000-dalton thylakoid membrane polypeptide.     

Rapid transient induction of phenylalanine ammonia-lyase mRNA in elicitor-treated bean cells

DNAs complementary to a size-selected fraction of poly(A)⁺ RNA present in elicitor-treated cells of bean (Phaseolus vulgaris L.) were inserted into pAT153 and used to transform Escherichia coli strain C600. Five clones were identified by hybrid-selected translation and cross-hybridization that contained sequences complementary to mRNA encoding phenylalanine ammonia-lyase (EC 4.3.1.5), which catalyzes the first reaction of phenylpropanoid biosynthesis. The longest insert contained a single open reading frame of 1520 base pairs together with 223 base pairs of 3′ untranslated sequence. RNA blot hybridization showed that elicitor caused a rapid, marked but transient increase in phenylalanine ammonia-lyase mRNA that was closely correlated with changes in translatable mRNA activity in vitro and enzyme synthesis in vivo. Blot hybridization of newly synthesized mRNA purified by organomercurial affinity chromatography following in vivo pulse-labeling with 4-thiouridine indicates that elicitor caused a rapid stimulation of phenylalanine ammonia-lyase mRNA synthesis as an early in the defense response leading to accumulation of phenylpropanoid-derived phytoalexins.     

Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.

Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2.     

Molecular cloning of mouse placental lactogen cDNA.

We have isolated a cDNA clone for the 23-kDa mouse placental lactogen II (mPL-II) from a phage lambda gt11 expression library containing cDNA synthesized from BALB/c placental RNA. Translation in vitro of placental mRNA selected by hybridization to the mPL-II cDNA clones yields a 26-kDa polypeptide that is the size of the expected precursor protein and that is immunoprecipitated with anti-mPL-II antiserum. The mPL-II cDNA clones hybridize to a 1.0-kilobase placental-specific mRNA. This mRNA, found in the fetal portion of the placenta, appears as early as day 10 of gestation and increases to a maximal level by day 12. The mPL-II cDNA nucleotide sequence has been determined. This sequence contains an open reading frame encoding a polypeptide of 222 amino acids with the amino-terminal 31 amino acids forming the signal sequence for secretion. The predicted secreted protein has 51% amino acid homology with mouse prolactin.