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.     

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.     

The mutant vasopressin gene from diabetes insipidus (Brattleboro) rats is transcribed but the message is not efficiently translated

The vasopressin gene from normal and diabetes insipidus (Brattleboro) rats has been isolated and sequenced. Except for a single deletion of a G residue in region coding for the neurophysin carrier protein the approximately 2300 nucleotides of both genes are identical. Blot analysis of hypothalamic RNA as well as transfection and microinjection experiments indicate that the mutant gene is correctly transcribed and spliced, however the resulting mRNA is not efficiently translated.     

Mechanisms of inherited deficiencies of multiple UDP-glucuronosyltransferase isoforms in two patients with Crigler-Najjar syndrome, type I.

Crigler-Najjar syndrome, type I (CN-I) is a potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from a recessively inherited deficiency of hepatic UDP-glucuronosyl-transferase (UGT) activity toward bilirubin (B-UGT). Two forms of B-UGT exist in human liver. mRNAs for these two forms and that for another isoform with activity toward simple phenols (P-UGT) have unique 5' regions, but their 3' regions are identical. The three mRNA species are derived from a single locus; the unique 5' regions are encoded by single unique exons and the identical 3' regions consist of four consecutive exons that are shared by all three isoforms. In this paper, we determined genetic lesions in two CN-I patients with deficiency of hepatic B-UGT and P-UGT activities. In one patient, there was a C----T substitution in exon 4 (common region) predicting the substitution of a serine residue with a phenylalanine residue; this mutation was present in the identical region of B-UGT and P-UGT mRNAs. In the other patient, a C----T substitution in exon 2 (common region) of the B-UGT/P-UGT locus resulted in a premature stop codon. This exon (132 nt) was absent in heptic B-UGT and P-UGT mRNAs of this patient due to exon skipping during pre-mRNA processing. Sequence abnormality of three distinct mRNA species explains the abnormality of multiple UGT isoforms in these patients. Presence of identical abnormalities in the common regions of the three mRNAs is consistent with the finding that the common 3' regions of the two B-UGT mRNAs and the P-UGT mRNA are encoded by four shared exons.     

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.     

The full length coding sequence of rat liver androsterone UDP-glucuronyltransferase cDNA and comparison with other members of this gene family.

Cloned cDNAs coding for hepatic UDP-glucuronyltransferase (UDPGT) have been isolated from a rat liver cDNA library in the expression vector bacteriophage lambda gt11 using anti-UDPGT antibodies. Four different mRNAs have been identified by sequencing of 15 UDPGT cDNA clones. The sequences of the four classes of cDNA were determined to be 85-95% homologous. Restriction fragments were isolated from the cDNA in each class and used as class specific probes. Hybridisation of these probes to northern blots of total RNA prepared from the livers of normal and genetically deficient Wistar rats identified the cDNA in class 4 with androsterone UDPGT. Translation of the cDNA sequence of clone rlug 23, the longest member of class 4, allowed determination of the complete amino acid sequence of androsterone UDPGT.     

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.     

Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA.     

Characterization of two types of histone H2B genes from macronuclei of Tetrahymena thermophila.

Two histone H2B gene clones were isolated from macronuclei of Tetrahymena thermophila. Nucleotide sequences of the two clones were highly homologous within the coding region but not in the noncoding region. Comparison of the deduced amino acid sequences between the two clones showed three differences in a total of 121 amino acids. Each of the two clones contained a TAA triplet within the coding region, which appeared to code for a glutamine residue. To demonstrate the existence of histone mRNA containing UAA triplet, nuclease P1 protection mapping using total cellular RNA and nucleotide sequencing of primer extension products were carried out. The results clearly indicated that two cloned histone H2B genes were transcribed, giving rise to the major histone H2B mRNAs with a UAA triplet sequence in frame. The tentative 5'- and 3'-ends of histone H2B mRNAs were determined.     

Construction of chimeric plasmids containing histone H5 cDNA from hen erythrocyte. DNA sequence of a fragment derived from the 5' region of H5 mRNA.

We report that construction and characterization of chicken erythrocyte histone H5 cDNA recombinant plasmids. cDNA was synthesized from poly(A)+ polysomal RNA enriched in H5 mRNA and inserted into the PstI site of pBR322. Several clones containing H5 cDNA sequences were obtained and one of them (p541), expressing H5 antigenic determinants, was sequenced. The DNA insert of p541 contains 118 nucleotides from the 5' non-translated region of H5 mRNA and sequences coding for up to residue 46 of the N-terminus of the arginine (position 15) H5 variant. There is a strikingly high number of repeated sequences both in the leader and coding region; among these, the octanucleotide 5' GCG GCG GC 3' is found five times along the sequence. Although the H5 mRNA 5' leader is GC-rich (66%), there is an AT-rich region, about 16 nucleotides long, which shares strong homology with the leaders of sea urchin histone H1 mRNAs.     

Distinct cis-acting signals enhance 3'' endpoint formation of CYC1 mRNA in the yeast Saccharomyces cerevisiae.

The cyc1-512 mutant of the yeast Saccharomyces cerevisiae contains a 38 bp deletion in the 3' untranslated region of the CYC1 gene, resulting in CYC1 mRNAs that are elongated, presumably labile, and reduced to 10% of the normal level. Analysis with S1 nuclease and a novel PCR procedure revealed that the low amount of cyc1-512 mRNA contained many discrete 3' termini at certain sites, ranging from the wild-type position to over 2000 nucleotides (nt) downstream. The cyc1-512 mRNA deficiency was completely or almost completely restored in eight intragenic revertants that contained six different single and multiple base-pair changes within a 300 bp region downstream from the translation terminator codon. Two of the six different reversions formed the sequence TAG...TATGTA, whereas the other four reversions created the sequences TATATA or TACATA. The positions of these revertant sequences varied, even though they caused an increased use of specific major downstream mRNA 3' endpoints, apparently identical to those seen in the cyc1-512 mRNA. However, several revertants contained minor end points not corresponding to any of the cyc1-512 mRNAs. The capacity of these three signals to form 3' ends was confirmed with sequences constructed by site-directed mutagenesis. We therefore suggest that the production of 3' termini of yeast mRNA may involve at least two functionally distinct elements working in concert. One type of element determines the sites of preferred 3' mRNA termini, as represented by the cyc1-512 termini. The second type of element, which includes TAG...TATGTA and TATATA motifs, operates at a distance to enhance the use of the downstream 3' preferred sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA.

Late SV40 16S and 19S mRNAs were found to contain an average of three m6A residues per mRNA molecule. The methylated residues of both the viral and cellular mRNAs occur in two sequences; Gpm6ApC and (Ap)nm6ApC, where n = 1-4. More than 60% of the m6A residues in SV40 16S and 19S mRNAs occur in Gpm6ApC even though there are twice as many (A)nAC than GAC sequences in these messengers. The m6A containing oligonucleotides of late SV40 MRNAs were localized in the viral messengers. In the 16S mRNA two m6A oligonucleotides were located at the 5' coding region between 0.95--0.0 map units. The third m6A residue was mapped between 0.0--0.14 map units in the translated portion of this mRNA. The overall pattern of internal methylation in the 19S mRNA is similar. However, some differences between 16S and 19S mRNAs were observed in both the content and location of the longer (Ap)n m6AC nucleotides. These results provide the first example of precise localization of internal methylation sequences in mRNA species with defined coding specificity. It implies that a) location of m6A residues is not random but specific to a particular region of the RNA, b) apart from sequence specificity other structural features of the mRNA may influence internal methylation and c) m6A residues are present in coding regions of SV40 mRNAs.