Large deletion of androsterone UDP-glucuronosyltransferase gene in the inherited deficient strain of Wistar rats

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5′-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.     

Large deletion of androsterone UDP-glucuronosyltransferase gene in the inherited deficient strain of Wistar rats.

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5'-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.     

Characterization and primary sequence of a human hepatic microsomal estriol UDPglucuronosyltransferase

A human liver microsomal UDP glucuronosyltransferase (UDPGT) that demonstrates reactivity with estriol (pI 7.4 UDPGT) has been purified to homogeneity and characterized further. No activity toward morphine, 4-hydroxybiphenyl, bilirubin, or tripelennamine was observed. The estriol UDPGT shows immunoreactivity with antibodies raised against rat hepatic microsomal 3 alpha- and 17 beta-hydroxysteroid UDPGTs but not with antibodies raised against rat hepatic microsomal p-nitrophenol UDPGT. The NH2-terminal sequence of the purified protein was determined and found to correspond to an identical sequence in the deduced amino acid sequence of a cDNA obtained from a human liver library in lambda gt11 (HLUG4). Sequence analysis revealed that HLUG4 is 2094 bp in length and encodes a protein of 523 amino acids which has a 16 amino acid leader sequence, followed by an untranslated 3' region of 525 bp. Three potential N-glycosylation sites were identified in the predicted sequence. The deduced amino acid sequence of estriol UDPGT showed 82% identity with the deduced amino acid sequence of another human hepatic cDNA (HLUG25), which has been expressed as a UDPGT capable of 6 alpha-hydroxyglucuronidation of hyodeoxycholic acid, strongly suggesting that these proteins are members of the same gene subfamily.     

The 3-methylcholanthrene-inducible UDP-glucuronosyltransferase deficiency in the hyperbilirubinemic rat (Gunn rat) is caused by a -1 frameshift mutation

The Gunn rat is a mutant strain of Wistar rat which has unconjugated hyperbilirubinemia as a result of the absence of hepatic UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. The Gunn rat is also deficient in a 3-methylcholanthrene (MC)-inducible UDPGT isoenzyme that has high activity toward phenolic substrates. We have isolated and sequenced a cDNA, designated 4-NP UDPGT, which encodes an MC-inducible UDPGT from normal Wistar rat livers (Iyanagi, T., Haniu, M., Sogawa, F., Fujii-Kuriyama, Y., Watanabe, S., Shively, J.E., and Anan, K.F. (1986) J. Biol. Chem. 261, 15607-15614). In the present study, we found that this cDNA detected MC-inducible UDPGT mRNA in the MC-treated homozygous Gunn rat liver. The level of this mRNA, however, was significantly lower than that of normal Wistar livers. The size of mRNA in Gunn rats was identical to that of the functionally mature UDPGT mRNA in Wistar rats, but the MC-inducible UDPGT protein was absent from homozygous Gunn rat microsomes. We therefore made a cDNA library from MC-treated Gunn rat liver mRNA and isolated cDNA clones, using the 4-NP UDPGT cDNA as a probe. Sequencing analysis of these cDNA clones revealed a single base deletion in the coding region. Northern blot analysis of mRNAs from normal Wistar and heterozygous and homozygous Gunn rats livers was performed using specific oligonucleotide probes, and the results confirmed the presence of mRNA containing the single base deletion in heterozygous and homozygous Gunn rats. These data suggested that the defect of the MC-inducible isoenzyme in Gunn rats arises from a -1 frameshift mutation that removes 115 amino acids from the COOH terminus.     

Analysis by molecular cloning of the human class II genes

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.     

Class II genes of miniature swine

Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.     

cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.     

Molecular basis of multiple UDP-glucuronosyltransferase isoenzyme deficiencies in the hyperbilirubinemic rat (Gunn rat).

The Gunn rat is a mutant strain of Wistar rat which has unconjugated hyperbilirubinemia as a result of the absence of hepatic UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. The Gunn rat is also deficient in UDPGT activities toward phenol substrates, and also toward digitoxigenin-monodigitoxiside. We have demonstrated that the defect of the isoenzyme for 4-nitrophenol (4NP) in Gunn rat liver arises from a -1 frameshift mutation that removes 115 amino acids from the COOH terminus (Iyanagi, T., Watanabe, T., and Uchiyama, Y. (1989) J. Biol. Chem. 264, 21302-21307). To investigate the molecular basis of defects in other UDPGT isoenzymes, we isolated and sequenced cDNAs from a Gunn rat liver library using mutant 4NP-UDPGT cDNA as a probe. Three novel cDNAs were identified that had identical 3'-regions of 1362 base pairs containing a single-base deletion in the same position as that of the mutant 4NP-UDPGT cDNA. However, their 5'-regions, encoding the substrate-binding domain, showed no more than 40% homology to that of 4NP-UDPGT. These data provide evidence that defects in some UDPGT isoenzymes in the Gunn rat are caused by a single mutation that results in the formation of a common truncated COOH terminus. Furthermore, the data also suggest that these mRNAs are transcribed from a single gene and that the 5'-exons are transcribed independently and differentially spliced to common 3'-exons encoding the conserved domain.     

Separation, purification and characterization of three isoenzymes of UDP-glucuronyltransferase from rat liver microsomes

Three isoenzymes of UDP-glucuronyltransferase (UDPGT) have been separated and purified from liver microsomes of untreated female rats or female rats pretreated with 3-methylcholanthrene. The UDPGT isoenzymes were purified utilizing Chromatofocusing, column isoelectric focusing, and UDP-hexanolamine Sepharose 4B affinity chromatography. UDPGT activities could also be separated during UDP-hexanolamine affinity chromatography by elution with different UDPGA (UDP-glucuronic acid) concentrations. One isoenzyme exhibits a subunit molecular weight of 56,000 and is capable of conjugating p-nitrophenol, 1-naphthol, and 4-methylumbelliferone. This isoenzyme is inducible by 3-methylcholanthrene treatment and requires high UDPGA concentrations for elution from the UDP-hexanolamine affinity column in contrast to the other UDPGT isoenzymes. A second isoenzyme was purified and displayed a subunit molecular weight of 50,000. This isoenzyme was not induced by 3-methylcholanthrene and was active towards testosterone, the 17-OH position of beta-estradiol, p-nitrophenol, and 1-naphthol. A third isoenzyme was also purified and exhibited a subunit molecular weight of 52,000. This isoenzyme conjugated androsterone and etiocholanolone and was not induced by 3-methylcholanthrene treatment. This study reports the purification of two separate and distinct rat liver UDPGT isoenzymes capable of conjugating p-nitrophenol, only one of which is inducible by 3-methylcholanthrene treatment. Also, this is the first report of the purification of a UDPGT isoenzyme active towards the 3-OH position of androgens.     

Isolation of a clone containing human histone genes.

A recombinant clone containing human histone genes has been isolated. The clone, lambda HH-01, was selected from a genomal library using chicken histone cDNA and a cloned fragment containing chicken histone genes as probes. Sub-clones from lambda HH-01 have been mapped and coding regions located with cDNA. The human H3 gene has been identified by DNA sequence analysis.     

Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain

Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.     

Cloning of cDNAs coding for rat hepatic microsomal UDP-glucuronyltransferases

Radioiodinated, affinity-purified, anti-UDP-glucuronyltransferase (UDPGT) antibodies have been used to isolate cDNAs coding for UDPGT(s) from a rat liver cDNA library cloned in the expression vector bacteriophage lambda gt11. The sizes of ten cloned cDNAs range from 0.3-2.1 kb. The identity of the cDNAs was confirmed by the hybrid-select translation and immunochemical analyses. Restriction mapping indicates that two classes of cDNA coding for different UDPGT mRNAs have been cloned.     

Linkage of a new member of the lectin supergene family to chicken Mhc genes

With the use of tissue-specific cDNA probes, several genes, which do not correspond to the class I (B-F), class II (B-L), or class IV (B-G) genes, were detected within the cosmid clusters containing the chicken major histocompatibility genes. We isolated cDNA clones with a probe corresponding to one of them, the 17.5 gene, located between two class I genes. The 17.5.3 cDNA, isolated from a chicken spleen cDNA library, encodes a 257-residue-long protein. This sequence shows significant similarity with several members of the C-type animal lectin superfamily and is probably a type II transmembrane protein. Analysis of several cDNA clones, together with Southern blot experiments, strongly suggest that this gene belongs to a multigene family, with at least some of its members being polymorphic. Several arguments lend support to the possibility that, together with the linked Mhc genes, the 17.5 gene is part of the recently described Rfp-Y system.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M88072.     

Human apolipoprotein A-II: complete nucleic acid sequence of preproapoA-II

The complete cDNA nucleic acid sequence of preproapolipoprotein (apo) A-II, a major protein constituent of high density lipoproteins, has been determined on clones from a human liver ds-cDNA library. Clones containing ds-cDNA for apoA-II were identified in the human liver ds-cDNA library using synthetic oligonucleotides as probes. Of 3200 clones screened, 4 reacted with the oligonucleotide probes. The DNA sequence coding for amino acids ?17 to +17 of apoA-II were determined by Maxam-Gilbert sequence analysis of restriction fragments isolated from one of these clones, pMDB2049. The remainder of the cDNA sequence was established by sequence analysis of a primer extension product synthesized utilizing a restriction fragment near the 5'-end of clone pMDB2049 as primer with total liver mRNA. The apoA-II mRNA encodes for a 100 amino acid protein, preproapoA-II that has an 18 amino acid prepeptide and a 5 amino acid propeptide terminating with a basic dipeptide (Arg-Arg) at the cleavage site to mature apoA-II.     

Complete nucleotide sequence of a unique HLA class I C locus product expressed on the human choriocarcinoma cell line BeWo

We have used Northern blot analysis to detect mRNA from class I HLA genes in the human choriocarcinoma cell line BeWo, which has been previously shown to express an atypical HLA class I molecule, in the absence of HLA A and B. Hybridization was seen with three class I DNA probes, the strongest being seen with the probe pC800, which corresponds to an 800-bp section of the Cw3 gene. We have made cDNA libraries from BeWo cells and screened for positive clones by using class I DNA probes. Of the clones isolated, we determined the complete sequence of one and partial sequence of five shorter clones. They all code for an identical C locus-related product, which does not correspond to published C locus sequences.     

Class II genes of miniature swine

Genomic clones corresponding to class II genes of theSLA ^c haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding theDRB andDQB genes were identified on the basis of hybridization with locus-specific 3 untranslated cDNA probes. Cluster 4 contained exons of bothDOB andDQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containingDP, DR, andDO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the association number M29944. Address correspondence and offprint requests to: C. LeGuern.     

Characterization of nodule-specific cDNA clones from Sesbania rostrata and expression of the corresponding genes during the initial stages of stem nodules and root nodules formation

We have constructed a Sesbania rostrata stem nodule-specific cDNA library. By screening with heterologous probes from pea and soybean, we have isolated several nodulin cDNA clones. On the basis of nucleotide and amino acid sequence homology, two nearly full-length cDNA clones coding for two different leghemoglobin-like proteins have been identified. The inserts of two other clones reveal a high degree of amino acid sequence homology (81% and 72%) to the early nodulin Enod2 from soybean; the characteristic heptapeptide repeat units PPHEKPP and PPYEKPP of the soybean Enod2 are conserved in the proteins encoded by these Sesbania cDNA clones. The time course of Enod2 and leghemoglobin mRNA appearance during the formation of stem nodules and root nodules on S. rostrata was analyzed by northern blot hybridization. Significant differences were found for the initiation of mRNA accumulation of these nodulins between S. rostrata and soybean.     

Brain 4-aminobutyrate aminotransferase. Isolation and sequence of a cDNA encoding the enzyme.

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It is responsible for the conversion of the neurotransmitter 4-aminobutyrate to succinic semialdehyde. By using oligonucleotide probes based on partial amino acid sequence data for the pig brain enzyme, several overlapping cDNA clones of 2.0-2.2 kilobases in length have been isolated. The largest cDNA clone was selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the precursor of 4-aminobutyrate aminotransferase consists of the mature enzyme of 473 amino acid residues and an amino-terminal segment of 27 amino acids attributed to the signal peptide. The cofactor pyridoxal-5-P is bound to lysine residue 330 of the deduced amino acid sequence of the mature enzyme.