Molecular cloning, sequencing, and expression of mouse ferrochelatase

The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody. Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained. These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions. From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692). The cDNA allows for the expression of active ferrochelatase by transfected culture cells. RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites. Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide. The band pattern of the RNA of the mouse liver was the same as that of the MEL cells. Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type.     

Kinesin mRNA Is Present in the Squid Giant Axon

Abstract: Recently, we reported the construction of a cDNA library encoding a heterogeneous population of polyadenylated mRNAs present in the squid giant axon. The nucleic acid sequencing of several randomly selected clones led to the identification of cDNAs encoding β-actin and β-tubulin, two relatively abundant axonal mRNA species. To continue characterization of this unique mRNA population, the axonal cDNA library was screened with a cDNA probe encoding the carboxy terminus of the squid kinesin heavy chain. The sequencing of several positive clones unambiguously identified axonal kinesin cDNA clones. The axonal localization of kinesin mRNA was subsequently verified by in situ hybridization histochemistry. In addition, the presence of kinesin RNA sequences in the axoplasmic polyribosome fraction was demonstrated using PCR methodology. In contrast to these findings, mRNA encoding the squid sodium channel was not detected in axoplasmic RNA, although these sequences were relatively abundant in the giant fiber lobe. Taken together, these findings demonstrate that kinesin mRNA is a component of a select group of mRNAs present in the squid giant axon, and suggest that kinesin may be synthesized locally in this model invertebrate motor neuron.     

Isolation and characterization of variant cDNAs encoding mouse tyrosinase

Two different cDNA clones encoding mouse tyrosinase (monophenol oxygenase, E.C. 1.14.18.1) were isolated from B16 melanoma cells, and their primary structure was determined. One of the cDNAs consists of 3309 nucleotides with an open reading frame coding for a peptide of 533 amino acids. The other cDNA is approximately 1600 nucleotides long, with a shorter 3'-untranslated region and a deduced in-frame deletion of 77 amino acid residues with respect to the former clone. Neither of these clones is structurally identical to other described mouse tyrosinase cDNAs (1-3). RNA blotting analysis demonstrates that multiple tyrosinase mRNA species are not only present in B16 melanoma, but also in normal skin melanocytes.     

Cloning,sequencing and expression of locust tropomyosin

Several different clones which contain sequences complementary to the mRNA encoding tropomyosin were isolated from cDNA libraries prepared from locust RNA. Based on the sequence analysis of available clones, the complete primary structure of the locust tropomyosin was explored. The deduced protein sequence showed a repeating pattern of amino acid residues characteristic of a coiled-coil structure. The amino acid sequence of locust tropomyosin contains domains of complete homology but also regions of pronounced variability when compared with tropomyosins of other species. Northern blot as well as Western blot analysis revealed that different forms of tropomyosins are expressed in locust muscles.     

Single base substitution between human intestinal and hepatic apolipoprotein B mRNA detected by ribonuclease cleavage analysis

The molecular mechanism of human intestinal apolipoprotein (apo) B-48 synthesis has been elucidated by a combination of sequencing of cloned complementary DNAs and RNase cleavage analysis of RNA heteroduplex. All intestinal cDNA clones contained a single C to T base substitution in the codon CAA encoding Gln2153 in apoB-100 cDNA, resulting in a translational stop. One of the our intestinal apoB cDNA clones was polyadenylated 106 bases downstream from the stop codon, possibly producing a 7-kb apoB message in the intestine. RNase cleavage analysis of the RNA heteroduplex between hepatic or intestinal RNA and apoB cDNA-directed anti-sense RNA showed that this single C to U substitution may occur in most of intestinal apoB mRNA. These results suggested that human apoB-48 is mostly produced by apoB mRNA with an in-frame stop codon in the intestine.     

Molecular cloning of DNA complementary to rat alpha 2-macroglobulin mRNA

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein synthesized in the liver. Using an in vitro translation system coupled with solid-phase radioimmunoassay, alpha 2M mRNA activity was found to rise to a maximum level in 16-24 h after turpentine injection. Poly(A)+ RNA from turpentine-injected rat liver was converted to cDNA by the method of Okayama-Berg, and about 50,000 transformants were obtained. From these transformants, clones containing alpha 2M cDNA were selected using the following criteria: 1) alpha 2M cDNA should hybridize with synthetic oligonucleotides encoding portions of the alpha 2M amino acid sequence, 2) alpha 2M cDNA should hybridize preferentially with RNA which increases during inflammation, 3) mRNA which hybridizes with alpha 2M cDNA should encode a polypeptide which specifically reacts with antibody against alpha 2M, and 4) the cDNA should contain the nucleotide sequences encoding the amino acid sequences of alpha 2M. We found clones which fulfilled these criteria. Using the cDNA clone as a probe, we demonstrated that the level of alpha 2M mRNA in the liver of inflamed animal markedly increased up to 1000-fold. The size of the alpha 2M mRNA was about 4800 nucleotides in length by Northern analysis.     

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.     

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.     

Molecular cloning and nucleotide sequence of cDNA encoding hydroxyindole O-methyltransferase of bovine pineal glands

Messenger RNA for hydroxyindole O-methyltransferase (EC 2.1.1.4) was partially purified from poly(A)+ RNA isolated from bovine pineal glands by sucrose density gradient centrifugation. The enriched mRNA was used to prepare a cDNA library by use of expression vector lambda gt11. The library was screened with monoclonal antibodies to the enzyme, and three cDNA clones were isolated. These cloned cDNAs cross-hybridized with one another, and their fusion proteins reacted to the monoclonal antibodies with different binding properties. Hydroxyindole O-methyltransferase enzymatic activity was demonstrated in the bacteria lysate infected with lambda HIOMT-A16, the clone that contained the longest insert. An almost full-length cDNA clone was isolated from lambda gt10 cDNA library by use of the lambda HIOMT-A16 cDNA as a probe. The primary structure of hydroxyindole O-methyltransferase was determined by analyzing the nucleotide sequence of the cDNAs. It consisted of 1939 nucleotides including a 1050-nucleotide region coding for 350 amino acids. RNA transfer blot analysis indicated that mRNA encoding hydroxyindole O-methyltransferase was present only in the pineal gland and not in the brain, retina, and liver of cow.     

Molecular cloning of hepatic mRNAs in Rana catesbeiana responsive to thyroid hormone during induced and spontaneous metamorphosis

Amphibian metamorphosis affords a useful experimental system in which to study thyroid hormone regulation of gene expression during postembryonic vertebrate development. In order to isolate gene-specific cDNA probes which correspond to thyroid hormone-responsive mRNAs, we employed differential colony hybridization of a cDNA library constructed from poly(A)+ RNA of thyroxine-treated premetamorphic tadpole liver. From an initial screening of about 6000 transformants, 32 "potentially positive" colonies were obtained. The recombinant cDNA-plasmids from 13 of these colonies plus two "potentially negative" colonies were purified for further study. Southern blot analysis of the plasmid DNA was employed to determine whether different cDNAs encoded for the same mRNA. The effect of thyroid hormone on the relative levels of specific mRNA species was examined by Northern analysis of liver RNA from premetamorphic tadpoles, thyroxine-treated tadpoles, and adult bullfrogs. Three independent cDNA clones were obtained which encoded thyroid hormone-enhanced mRNAs. We also obtained two independent cDNA clones encoding thyroid hormone-inhibited mRNAs and three independent clones encoding thyroid hormone-unresponsive mRNAs. The levels of two thyroid hormone-enhanced mRNAs and one thyroid hormone-inhibited mRNA were essentially the same in the thyroid hormone-treated tadpole liver and adult liver, suggesting that thyroid hormone induces stable changes in liver gene expression during spontaneous metamorphosis. Using selected cDNAs, RNA dot blot analysis of liver mRNA from tadpoles at different stages of metamorphosis showed that the level of one thyroid hormone-enhanced mRNA increased during late prometamorphosis and metamorphic climax. Similarly, a mRNA which was strongly inhibited by thyroid hormone treatment was observed to decline during prometamorphosis and reach undetectable levels during metamorphic climax. One mRNA was detected which was reproducibly inhibited by thyroid hormone treatment but which remained essentially unchanged during spontaneous metamorphosis. These results provide the first direct evidence for the coordinate and selective pretranslational regulation by thyroid hormone of several liver genes during the developmental process of metamorphosis.