Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Molecular cloning of cDNA for rat acyl-CoA oxidase

Poly(A+) RNA was prepared from hepatic free polysomes of rats which had been fed di(2-ethylhexyl) phthalate for the induction of peroxisomal beta-oxidation enzymes. This preparation was enriched for the mRNAs of these enzymes by sucrose density gradient centrifugation, and used for the synthesis of double-stranded cDNA. Recombinant plasmids were constructed from the cDNA and pBR322 by dG X dC-tailing method and used for the transformation of an Escherichia coli strain, chi 1776. By differential colony hybridization using [32P]cDNA of partially purified liver poly(A+) RNA from induced and noninduced rats as probes, and then by hybridization-selected translation, we obtained two clones with cDNA inserts which specifically selected acyl-CoA oxidase mRNA. On Northern blotting, both cDNA inserts hybridized to 3.8-kilobase RNA which was increased about 10-fold by di(2-ethylhexyl) phthalate treatment of the rats. The cleavage maps of the cDNA inserts showed they overlap with each other. We conclude that the above two recombinant plasmid clones contain cDNA sequences for rat acyl-CoA oxidase.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.     

Cloning and sequencing of the cDNA for S-acyl fatty acid synthase thioesterase from the uropygial gland of mallard duck

In vitro translation of poly(A)+ RNA from the uropygial glands of mallard ducks (Anas platyrhynchos) generated a 29-kDa protein which cross-reacted with rabbit antibodies prepared against S-acyl fatty acid synthase thioesterase (Kolattukudy, P. E., Rogers, L., and Flurkey, W. (1985) J. Biol. Chem., 260, 10789-10793). A poly(A)+ RNA fraction enriched in this thioesterase mRNA, isolated by sucrose density gradient centrifugation, was used to prepare cDNA which was cloned in Escherichia coli using the plasmid pUC9. Using hybrid-selected translation and colony hybridization, 17 clones were selected which contained the cDNA for S-acyl fatty acid synthase thioesterase. Northern blot analysis showed that the mature mRNA for this thioesterase contained 1350 nucleotides whereas the cloned cDNA inserts contained 1150-1200 base pairs. Five of the 6 clones tested for 5'-sequence had identical sequences, and the three tested for 3'-end showed the same sequence with poly(A) tails. Two clones, pTE1 and pTE3, representing nearly the full length of mRNA, were selected for sequencing. Maxam-Gilbert and Sanger dideoxy chain termination methods were used on the cloned cDNA and on restriction fragments subcloned in M13 in order to determine the complete nucleotide sequence of the cloned cDNA. The nucleotide sequence showed an open reading frame coding for a peptide of 28.8 kDa. Two peptides isolated from the tryptic digest of the thioesterase purified from the gland showed amino acid sequences which matched with two segments of the sequence deduced from the nucleotide sequence. Another segment containing a serine residue showed an amino acid sequence homologous to the active serine-containing segment of the thioesterase domain of fatty acid synthase. Thus, the clones represent cDNA for S-acyl fatty acid synthase thioesterase. The present results constitute the first case of a complete sequence of a thioesterase.     

Characterization of complementary DNA clones coding for two forms of 3-methylcholanthrene-inducible rat liver cytochrome P-450

Double-stranded DNA complementary to the partially purified mRNA prepared from 3-methylcholanthrene (MC)-treated rat liver was constructed and cloned in Escherichia coli. Twenty clones were verified to carry a complementary DNA (cDNA) insert coding for MC-inducible cytochrome P-450 by positive hybridization translation assay and immunochemical assay with anti-cytochrome P-450 antibody. The identified cDNA clones were divided into at least two groups on the basis of comparison of restriction maps of the cDNA inserts. A clone pAU157 whose cDNA insert was approximately 2.7 kb in length contained nearly full-length mRNA information for cytochrome P-450MC or P-450c, which is the major form of MC-inducible cytochrome P-450. Other cDNA clones pTZ286-pTZ330 contained the 1.2 kb sequence complementary to cytochrome P-450d mRNA. RNA blot analysis revealed that pAU157 and pTZ286-pTZ330 cDNA clones were derived from 22S and 18S mRNAs, respectively, both of which were induced in rat liver by MC treatment. Sequence analysis revealed that there were closely homologous sequence regions in pAU157 and pTZ286-pTZ330 cDNA inserts and most of the homologous sequences were localized in two limited coding regions of the two cytochrome P-450 species. pAU157 encoded the total amino acid sequence of cytochrome P-450MC or P-450c and pTZ286-pTZ330 coded for the C-terminal 368 amino acid residues of cytochrome P-450d. Two highly homologous regions were found in the amino acid sequences of these cytochrome P-450 species.     

Cloning of epoxide hydratase complementary DNA

Tightly membrane-bound polysomes were isolated from livers of rats administered trans-stilbene oxide. Epoxide hydratase mRNA was enriched from these polysomes using immunochemical techniques and oligo(dT)-cellulose chromatography. This resulted in an increase in message concentration over that found in noninduced membrane-bound cDNA, synthesized from enriched mRNA, was inserted into the ampicillin resistance gene of pBR322 using oligo(dG)-oligo(dC) tailing. Clones containing sequences complementary to epoxide hydratase mRNA were selected by differential colony hybridization using [32P]cDNA synthesized from immunoenriched mRNA and [32P]cDNA synthesized from nonenriched mRNA. Plasmids from four clones, which only annealed with the enriched probe, were isolated and shown to specifically hybridize with epoxide hydratase mRNA by hybrid selection-translation. A composite restriction endonuclease map of the plasmid inserts was constructed which spanned 1310 base pairs and represented approximately 80% of the message sequence. The 3'-5' orientation of this map relative to the epoxide hydratase mRNA was also determined.     

Molecular cloning of cDNA for rat mitochondrial 3-oxoacyl-CoA thiolase

Messenger RNA of rat 3-oxoacyl-CoA thiolase (acetyl-CoA acyltransferase), a mitochondrial matrix enzyme involved in fatty acid beta-oxidation, was enriched by immunoprecipitation of rat liver free polysomes and recombinant plasmids were prepared from the enriched mRNA by a modification of the vector-primer method of Okayama and Berg. The transformants were initially screened for 3-oxoacyl-CoA thiolase cDNA sequences by differential colony hybridization with [32P]cDNAs, synthesized from the immunopurified and unpurified mRNAs. The cDNA clones for 3-oxoacyl-CoA thiolase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pT1-1, contained a 700-base insert and hybridized to a mRNA species of 1.6 X 10(3) bases in rat liver. The transformants were rescreened using the cDNA insert of pT1-1 as a hybridization probe and a clone (pT1-19) with a 1.5 X 10(3)-base insert was obtained. Activity and concentration of 3-oxoacyl-CoA thiolase mRNA were quantified by in vitro translation and dot-blot analysis using the cDNA insert as a hybridization probe. The level of translatable and hybridizable mRNA in rat liver was increased about 5.1-fold and 4.6-fold, respectively, after administration of di-(2-ethylhexyl)phthalate, a potent inducer of the enzyme. The 3-oxoacyl-CoA thiolase mRNA levels thus determined correlated closely with levels of the activity and amount of this enzyme.     

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.     

Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA

A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.     

Induction by cAMP of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken. Identification and characterization of a cDNA clone for the enzyme

Previous work from our laboratory (Hod, Y., Utter, M. F., and Hanson, R. W. (1982) J. Biol. Chem. 257, 13787-13794) has demonstrated that chicken kidney contains both mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that the two forms are distinct proteins. Using poly(A+) RNA from chicken kidney, a double-stranded cDNA library was constructed. DNA clones containing sequences complementary to the mRNA for the cytosolic form of phosphoenolpyruvate carboxykinase were initially identified by colony hybridization with 32P-labeled cDNA transcribed from an RNA fraction enriched for the enzyme mRNA. The identity of plasmids containing phosphoenolpyruvate carboxykinase cDNA was confirmed by hybrid-selected translation. Mature mRNA for cytosolic phosphoenolpyruvate carboxykinase of the chicken is 2.8 kilobases in length, similar to that previously noted for mRNA coding for the same enzyme in the rat. The cDNA for the chicken enzyme hybridizes with several restriction fragments of the corresponding cDNA for the rat cytosolic phosphoenolpyruvate carboxykinase, indicating conservation of nucleotide sequences during evolution. Wide spread conservation of sequence homology is also demonstrated by the hybridization of the cDNA for the rat phosphoenolpyruvate carboxykinase with a 2.8-kilobase RNA from the livers of a variety of vertebrates including amphibian, avian, and primate species. Specific mRNA coding for the cytosolic form of phosphoenolpyruvate carboxykinase was present in chicken kidney but absent from the liver, even in animals starved for 48 h. However, the administration of cAMP to normal fed chickens caused a rapid induction of phosphoenolpyruvate carboxykinase mRNA. These findings suggest that the gene for the cytosolic enzyme in chicken liver can be expressed if the proper hormonal stimuli are present.     

Molecular cloning of cDNA for rat L-type pyruvate kinase and aldolase B

Two double-stranded cDNA recombinant pBR322 plasmid libraries were constructed starting from high carbohydrate diet rat liver poly(A)+ mRNA, either fractionated by denaturing sucrose gradient centrifugation for the cloning of L-type pyruvate kinase cDNA, or nonfractionated for aldolase B. Both libraries were screened with single-stranded cDNA probes reverse transcribed from fasted or high carbohydrate diet rat liver mRNAs. mRNAs from fasted animals were also fractionated by sucrose gradient centrifugation and mRNAs from the fed animals were, in addition, further purified by high performance liquid gel filtration chromatography. Those clones hybridizing with the "positive" probe (from animals fed the high carbohydrate diet) and not with the "negative" one (from fasted animals) were preselected and their plasmid DNA was purified and analyzed by positive hybridization-selection. Thirty of 4500 bacteria colonies transformed by recombinant plasmids were preselected by differential screening for pyruvate kinase, and 8 of 864 colonies for aldolase B. Twenty-two recombinant plasmids for pyruvate kinase and two for aldolase B were shown to contain specific cDNA inserts by positive hybridization-selection. Plasmids DNAs of some pyruvate kinase and aldolase B clones (whose inserts ranged from 700 to 1050 bases in length) were labeled by nick translation and used as probes for Northern blot hybridization. The pyruvate kinase cDNA probes recognized mainly a 3400-base RNA species which was detected in high carbohydrate diet rat liver, but not in fasted rat liver and in tissues which do not synthesize L-type pyruvate kinase. In addition, some pyruvate kinase probes hybridized with minor RNA species of about 2000 bases in length, only observed after carbohydrate diet. For aldolase B, the recombinant plasmid DNA hybridized with a single RNA species of 1750 bases. This RNA, detected in kidney, small intestine and liver, was induced by a high carbohydrate diet and increased with liver development. The rat probe cross-hybridized with human aldolase B messenger RNA.     

Molecular cloning of cDNA for rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase

Messenger RNA for 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial matrix enzyme of fatty acid beta-oxidation, was purified from livers of di(2-ethylhexyl)phthalate-treated rats by immunoadsorption of hepatic free polysomes to fixed cells of Staphylococcus aureus and enrichment for poly(A)-rich RNA by oligo(dT)-cellulose chromatography. Plasmid cDNA was constructed from this poly(A)-rich RNA by a modification of the method of Okayama and Berg and was transformed into the Escherichia coli DH1 strain. Plasmids containing cDNA sequences coding for 3-hydroxyacyl-CoA dehydrogenase were screened by differential colony hybridization, and were identified by hybrid-arrested translation and hybrid-selected translation. Plasmid pHADH-1, which contains a 1400-base-pair insert, hybridized to rat 3-hydroxyacyl-CoA dehydrogenase mRNA with a length of 1700 bases. Determination of the dehydrogenase mRNA by in vitro translation and dot-blot analysis with the cDNA probe showed that the induction of the enzyme in rat liver by di(2-ethylhexyl)phthalate could be attributed to an increase in the mRNA concentration.     

Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

Poly(A)+ RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A)+ RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from approximately 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G X C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH.     

Identification of recombinant phages containing sequences from different rat myosin heavy chain genes.

The construction and identification of a recombinant plasmid containing a cDNA insert which hybridizes specifically to myosin heavy chain mRNA is described. The plasmid was used as a probe to screen a rat genomic library for recombinant phages containing myosin heavy chain sequences. Six clones with approximately 15 k bp inserts each were isolated. Digestion with several restriction enzymes and hybridization of the fractionated DNA with the plasmid probe showed that the clones contained 3 different DNA inserts. Electron microscopy of a heteroduplex made by hybridization of DNA from two clones confirmed that the inserts originated in different genes. Hybridization of size-fractionated ECOR1 digested rat spleen DNA with the cloned probe suggested the existence of at least 5 myosin heavy chain genes.     

Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families

A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stage-specific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.     

Properties of rat and mouse beta-glucuronidase mRNA and cDNA, including evidence for sequence polymorphism and genetic regulation of mRNA levels

cDNA clones containing partial sequences for beta-glucuronidase (beta G) were constructed from rat preputial gland RNA and identified by their ability to selectively hybridize beta G mRNA. One such rat clone was used to isolate several cross-hybridizing clones from a mouse-cDNA library prepared from kidney RNA from androgen-treated animals. Together, the set of mouse clones spans about 2.0 kb of the 2.6-kb beta G mRNA. Using these cDNA clones as probes, a genomic polymorphism for DNA restriction fragment size was found that proved to be genetically linked to the beta G gene complex. A fragment of beta G cDNA was subcloned into a vector carrying an SP6 polymerase promoter to provide a template for the in vitro synthesis of single-stranded RNA complementary to beta G mRNA. This provided an extremely sensitive probe for the assay of beta G mRNA sequences. Using either nick-translated cDNA or transcribed RNA as a hybridization probe, we found that mouse beta G RNA levels are strongly induced by testosterone, and that induction by testosterone is pituitary-dependent. During the lag period preceding induction, during the induction period itself, and during deinduction following removal of testosterone, beta G mRNA levels paralleled rates of beta G synthesis previously measured by in vivo pulse-labelling experiments. Genetic variation in the extent of induction affected either the level of beta G mRNA or its efficiency of translation depending on the strain of mice tested.