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D Beier

Publications and source records attributed to D Beier.

At least 37 records · Page 2Linked to original sources
PubMed1998

[Polymerase chain reaction (PCR) for detection of BLV provirus-- a practical complement for BLV diagnosis?].

A typical infection with bovine leukemia virus (BLV) induces a permanent antibody (Ab) response with high titers against BLV-antigens. In the last few years atypical courses of infection with low or transient BLV-Ab-titers or even lack of any detectable BLV-Ab-titers in animals with BLV-provirus integration have been described. This makes it difficult to eliminate BLV infection from herds using serological assays only. Whether or not polymerase chain reaction (PCR) is a useful tool to complement serological Ab-assays in BLV-eradication in herds was clarified in three ways: (i) different DNA-quick-preparations of blood were examined in nested PCR, (ii) cows of a BLV infected herd that was involved in a national eradication program were investigated for 6 months und (iii) BLV-provirus-variants occurring in this herd were differentiated. The results show, that even by using PCR it was not possible to detect all infected animals all the time and that eradication of BLV from this herd was not completed in this short time. The PCR is useful for the investigation of herds and more sensitive than ELISA. PCR using LTR-primers (34 positive cattle) was more sensitive than PCR with env-primers (30 positive cattle). Using PCR 34 BLV infected cattle were detected of which only 21 reacted in ELISA. Restriction enzyme analysis or sequence analysis of PCR-amplificates allowed the detection of virus variants and conclusions about the way of infection. PCR should be used for BLV-eradication in cattle herds with low BLV-incidence, for the investigation of new outbreaks or tumor cases in long term BLV free herds and for investigation of breeding cattle.

Amino Acid Sequence↗
PubMed1998

[Possibilities and limitations for use of the polymerase chain reaction (PCR) in the diagnosis of bovine leukemia virus (BLV) infection in cattle].

Enzootic bovine leukosis is caused by the bovine leukemia virus (BLV) and has a world wide distribution in cattle. Due to the program for eradication of BLV-infections in Germany the BLV incidence in cattle declined and only few new cases seem to occur per year. On the other hand, BLV-infected cattle with low, transient or without BLV-antibody titers are difficult to identify as BLV-infected. These animals may be sources for new infections. It was the aim of this study to compare the suitability of agargel-immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) for diagnosis of BLV-infected cattle. We investigated a herd with 10 cows, where after a long period when the herd was negative suddenly a positive serological reaction appeared. In addition 64 animals from 6 federal states of different herds with doubtful serological reactions found in previous tests were included. In the herd with 10 cows we were able to detect BLV-infection in one animal 8 weeks earlier with PCR than with ELISA. Investigation of 56 adult cattle and 3 calves from different herds with both PCR and ELISA showed that 51 animals were positive in ELISA and 55 in PCR. Seven animal were positive in PCR and negative in ELISA. Three calves yielded negative results in PCR and positive results in ELISA. One cow which was positive in previous serological tests was negative in ELISA, AGID and PCR. Restriction fragment length polymorphism analysis demonstrated that the majority of the cattle was infected with the same BLV provirus variant. The four PCR variants used in this study yielded a similar sensitivity for BLV provirus detection. In conclusion, compared to the serological tests, PCR detects BLV-infection earlier in naturally infected cattle. The method is also a useful tool to exclude or confirm BLV-infection in cattle with doubtful serological results. PCR may be used to complement the serological tests in the diagnosis of BLV-infection.

Animals↗
PubMed1997

Provirus variants of the bovine leukemia virus and their relation to the serological status of naturally infected cattle.

Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of the env gene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5. 3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.

Amino Acid Sequence↗
PubMed1997

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

Alleles↗
PubMed1997

Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori.

Helicobacter pylori strains isolated from most patients with peptic ulcer disease and adenocarcinoma express the vacuolating toxin VacA and contain a pathogenicity island named cag. The cag pathogenicity island codes for more than 40 putative proteins with features similar to bacterial secretion systems. One of these proteins, CagA, is an immunodominant antigen with unknown function encoded by the cagA gene. In the present study, we have analysed the functional promoter elements of the H. pylori cagA gene as well as of the divergently transcribed cagB gene. Primer extension analyses identified a single 5' end of the cagA mRNA, while two initiation sites were mapped in the case of the cagB mRNA. The promoters deduced upstream of these start points of transcription contained conserved -10 regions but no -35 regions with respect to the Escherichia coli sigma70 consensus sequence. Nevertheless, they could be activated in E. coli and in vitro by purified E. coli RNA polymerase. Deletion analyses indicated that the cagA and cagB genes are transcribed by overlapping promoters and that full activation requires sequences up to -70 and -96 respectively. Instead, basal transcription is likely to be mediated by -10 extended promoter-like sequences. RNA polymerase is able to bind the -40 to -60 region of the cagA promoter, and its binding is mediated by the alpha-subunit. This region resembles the UP elements of prokaryotic promoters in location, sequence and mechanism of interaction with the RNA polymerase. We discuss the features of these promoters and propose that they could represent a class of minimum promoters, which ensures a basic level of transcription, while full activation requires regulatory elements or a defined promoter context.

Antigens, Bacterial↗
PubMed1997

Identification and characterization of an operon of Helicobacter pylori that is involved in motility and stress adaptation.

We identified a novel stress-responsive operon (sro) of Helicobacter pylori that contains seven genes which are likely to be involved in cellular functions as diverse as chemotaxis, heat shock response, ion transport, and posttranslational protein modification. The products of three of these genes show amino acid homologies to known proteins, such as the flagellar motor switch protein CheY, a class of heat shock proteins, and the ribosomal protein L11 methyltransferase, and to a phosphatidyltransferase. In addition to containing an open reading frame of unknown function, the product of which is predicted to be membrane associated, the sro locus contains three open reading frames that have previously been described as constituting two separate loci, the ftsH gene and the copAP operon of H. pylori. Knockout mutants showed that CheY is essential for bacterial motility and that CopA, but not CopP, relieves copper toxicity. Transcriptional analyses indicated that this locus is regulated by a single promoter and that a positive effect on transcription is exerted by the addition of copper to the medium and by temperature upshift from 37 to 45 degrees C. The possible role of this locus in H. pylori virulence is discussed.

Adaptation, Physiological↗
PubMed1996

Conserved sequence motifs in the unorthodox BvgS two-component sensor protein of Bordetella pertussis.

The unorthodox two-component sensor protein BvgS of Bordetella pertussis contains several interesting sequence motifs of unknown functional relevance, such as a histidine motif in its output domain, which is conserved among several unorthodox sensor proteins, a putative nucleotide binding site [Walker box type A] in its linker region, and a region in its periplasmic domain with significant homology to the TonB protein of Escherichia coli. We investigated potential functions of these sequences by constructing B. pertussis strains that express mutant BvgS derivatives. The His1172 residue in the output domain was exchanged for Gln, and the Walker motif was mutated either by the replacement of Lys625 by Arg, or of Gly624 by Val and Lys625 by Leu. To analyse the TonB motif, the periplasmic domain of BvgS was replaced with the corresponding domain of EvgS, an E. coli sensor that is highly homologous to BvgS but lacks the similarity with TonB. All mutations except the conservative Lys/Arg exchange in the Walker box caused the inactivation of BvgS, indicating the functional importance of the conserved motifs. The activity of the mutant proteins could be restored by complementation in trans with various separately expressed, truncated parts of BvgS. Mutations in the BvgS receiver domain could be complemented not only by a construct expressing the wild-type receiver and output domains, but also by the derivative containing the His-Gln exchange. Therefore, the histidine motif, although important for BvgS function, is not essential for complementation of BvgS mutants. The mutations in the Walker box and in the periplasmic domain could be complemented by a truncated BvgS derivative lacking the receiver and output domains. The characterization of a spontaneous revertant of the strain expressing the originally inactive EvgS/BvgS hybrid protein revealed the presence of a mutation in the BvgS linker region, conferring constitutive activity on the protein. As TonB energizes transport processes across the outer membrane of E. coli, the strain expressing the constitutive EvgS/BvgS hybrid protein lacking the TonB motif was used in preliminary investigations of a possible direct involvement of BvgS in transport processes.

Amino Acid Sequence↗
PubMed1996

Regulation of hepatocyte bile salt transporters by endotoxin and inflammatory cytokines in rodents.

BACKGROUND & AIMS: Pathophysiological conditions such as sepsis and hepatitis are mediated by inflammatory cytokines and frequently are associated with cholestasis. The aim of this study was to determine the effect of endotoxin (lipopolysaccharide [LPS]) and cytokine administration on hepatocellular transporters involved in bile salt transport. METHODS: LPS and cytokines were administered to Sprague-Dawley rats or C57BL/6 mice, and the expression and function of hepatocyte transporters involved in bile salt secretion were examined. RESULTS: LPS caused gene expression of the hepatocyte basolateral sodium-dependent taurocholate cotransporter (Ntcp) to decrease by more than 90%. Tumor necrosis factor alpha (TNF-alpha) or interleukin (IL) 1beta also produced a time-dependent decrease in Ntcp messenger RNA levels, whereas IL-6 had no effect. LPS administration resulted in a concordant 90% reduction of basolateral protein expression of the hepatocyte sodium taurocholate cotransporter and markedly diminished sodium-dependent taurocholate uptake. Activity of the hepatocyte basolateral Na+,K+-adenosine triphosphatase (ATPase) was also decreased by 50% in a posttranslational manner after endotoxin treatment. CONCLUSIONS: Endotoxin inhibits hepatocellular sodium-dependent bile salt uptake by decreasing both expression of Ntcp and activity of the Na+,K-ATPase. The effects on Ntcp are mediated via TNF-alpha and IL-1beta. Alterations of these transporters may contribute to the cholestasis of sepsis and inflammation.

Animals↗
PubMed1996

Evaluation of polymerase chain reaction (PCR) application in diagnosis of bovine leukaemia virus (BLV) infection in naturally infected cattle.

The practical application of polymerase chain reaction (PCR) for the diagnosis of bovine leukaemia virus (BLV) infections in naturally infected cattle was evaluated. Compared to serological tests the PCR was definitely found to be a more sensitive method, yielding the highest number of positive results (10% more compared to enzyme-linked immunosorbent assay, (ELISA), and 17.7% more compared to agar-gel immunodiffusion, (AGID)). In testing cattle from herds with BLV incidence under 5%, out of 52 provirus positive cattle only 43 were correctly identified by ELISA. When compared to AGID only 37 of the 52 PCR positive animals were correctly identified. Of 18 cattle imported from the Slovak Republic and kept in a quarantine stable, four were found to be BLV provirus positive by PCR, while serological tests indicated one animal positive and three negative. Therefore, it is impossible to prevent the spread of the infection from one country to another by serological testing only. Moreover, it is feasible to identify animals with changing antibody titres correctly by PCR. Using PCR we were also able to distinguish BLV infected from uninfected calves that were serologically positive due to colostral antibodies. Higher sensitivity of BLV provirus detection by PCR was achieved using env gene rather than tax gene specific primers. Negative results by PCR in cases of positive serological reactions are still possible, as shown in case of one adult animal. These findings indicate that PCR is a highly sensitive method and might be successfully used and economically advantageous for different practical applications in detection of BLV infection in naturally infected cattle.

Animals↗
PubMed1996

Signal transduction and virulence regulation in Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, coordinately regulates the expression of its virulence factors in response to certain environmental stimuli. This coordinate regulation is accomplished by the bvg locus encoding the BvgS and the BvgA proteins, which are members of the two-component family of bacterial signal transducing proteins. The sensor protein BvgS shows an "unorthodox" domain structure, combining the characteristic communication modules both of the two component sensors and response regulators, each of which is indispensable for BvgS function. Although under global control of the BvgAS system, two subsets of virulence factors exemplified by the adhesin FHA and the toxins PTX and CYA exhibit, respectively, a differential mode of expression. This is reflected in a differential kinetics of transcriptional activation in vivo, and the different ability of the various virulence promoters to be expressed in the heterologous organism Escherichia coli. Evidence is accumulating that this differential regulation may be due to different affinities of the virulence promoters for the phosphorylated form of BvgA.

Bacterial Adhesion↗
PubMed1995

In vivo characterization of the unorthodox BvgS two-component sensor protein of Bordetella pertussis.

Two-component sensor proteins are typically composed of an amino-acid sensory and a carboxy-terminal transmitter domain containing a kinase activity which catalyses the autophosphorylation of a histidine residue. In a second step, the phosphate is transferred to aspartic acid residues located in the receiver domain of the second component, the response regulator. A few sensor proteins such as the BvgS protein of Bordetella pertussis have a more complex structure. BvgS possesses additional C-terminal domains, including receiver and output modules usually found only in the response regulators. The function of these BvgS domains was investigated by mutation and complementation analysis in vivo. BvgS derivatives were constructed lacking the C-terminal domains or containing mutations in conserved amino acids. All mutations caused the inactivation of BvgS as measured by the expression of virulence factors at the transcriptional and translational level after integration of the mutated alleles in the B. pertussis chromosome. However, some of these mutants could be complemented to the wild-type phenotype by the separate expression of various C-terminal BvgS domains in trans indicating a direct interaction of the truncated and complete BvgS proteins. Therefore, the dimerization capacity of the cytoplasmic BvgS domains was analysed using a lambda repressor based dimerization probe system. These results indicated that BvgS has two dimerization regions, one in the transmitter and a second in the C-terminal receiver/output domains. Furthermore, several BvgS hybrid proteins were constructed which contained substitutions of the BvgS receiver and output domains with similar domains of two-component response regulators and of the sensor protein EvgS. It was found that the receiver domain does not carry BvgS-specific functions and can be exchanged by a heterologous receiver domain. However, the BvgS output domain could not be substituted with output domains of the related proteins without inactivation of BvgS.

Amino Acid Sequence↗
PubMed1994

Interleukin-6 inhibits hepatocyte taurocholate uptake and sodium-potassium-adenosinetriphosphatase activity.

The potential effects of cytokines on hepatocellular transport functions remain undefined. Interleukin-6 (IL-6) is a cytokine that is produced in sepsis, hepatitis, and other inflammatory conditions often associated with cholestasis. Using cultured rat hepatocytes, we have investigated the effects of IL-6 on hepatocellular bile salt uptake. Because hepatocyte Na(+)-K(+)-adenosinetriphosphatase (ATPase) produces the electrochemical gradient that drives sodium-dependent bile salt contransport, we also examined the effects of IL-6 on Na(+)-K(+)-ATPase activity. Hepatocytes cultured for 20 h in media containing IL-6 exhibited a dose-dependent noncompetitive inhibition of [3H]taurocholate uptake, which was maximal at an IL-6 dose of 100 U/ml. IL-6 treatment had no effect on hepatocyte sodium-independent taurocholate uptake. Northern blotting of RNA from cultured hepatocytes revealed that IL-6 had no effect on steady-state RNA levels of the Na(+)-taurocholate transporter (Ntcp). Hepatocytes incubated with IL-6 for 20 h, however, exhibited a 55% decrease in hepatocyte Na(+)-K(+)-ATPase activity. This effect also was dose dependent, with maximal inhibition occurring at an IL-6 dose of 100 U/ml. Similar treatment with IL-6 did not influence hepatocyte Mg(2+)-ATPase activity. The inhibition of Na(+)-K(+)-ATPase activity induced by IL-6 provides a putative mechanism for the observed inhibition of sodium-dependent taurocholate uptake. Since modulation of bile salt transport and Na(+)-K(+)-ATPase activity occurred at IL-6 concentrations comparable to the serum levels observed in patients with severe inflammatory states, these findings have potential pathophysiological relevance for the cholestasis of sepsis and other inflammatory disorders.

Animals↗
PubMed1994

[Prevalence of antibodies against the viruses of European swine fever, Aujeszky's disease and "porcine reproductive and respiratory syndrome" in wild boars in the federal states Sachsen-Anhalt and Brandenburg].

During the hunting season from 1991/1992 blood samples were collected from wild boar shot in the Federal States of Sachsen-Anhalt (482 samples) and Brandenburg (177 samples) which corresponds to 2.1 and 0.4% of the total hunting bag. All sera were screened in a complex trapping blocking (CTB) ELISA for antibodies against hog cholera virus (HCV) and in an indirect ELISA for antibodies against Aujeszky's disease virus (ADV). Additionally the sera were tested for neutralizing antibodies against HCV strain ALFORT/187, bovine viral diarrhoea virus (BVDV) strains NADL and 1138/69, and against an ADV field isolate. In case of questionable results sera were tested against HCV strain "BERGEN", HCV vaccine strain "RIEMS" and three HCV field isolates from wild boar. The serological testing for antibodies against "porcine reproductive and respiratory syndrome virus" (PRRSV) was carried out in indirect immunoperoxidase monolayer assay (IPMA). Four sera (Sachsen-Anhalt) reacted positive in CTB-ELISA. Seven sera yielded neutralizing antibodies against HCV but only one of the "non-negative" samples scored positive in both techniques, ELISA and VNT. Two sera (Brandenburg) had low neutralizing antibody titres against Alfort/197 but scored negative in CTB-ELISA. Screening for antibodies against ADV of 640 sera led to 13 positive sera including 5 positive findings in both ELISA and VNT. Antibodies against PRRSV were detected in two sera which were collected Sachsen-Anhalt. Estimations resulted in a prevalence of about 5% for antibodies against HCV.

Animals↗
PubMed1992

Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain.

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.

Arabidopsis↗
PubMed1992

The tRNA(Tyr) multigene family of Nicotiana rustica: genome organization, sequence analyses and expression in vitro.

Tobacco tRNA(Tyr) genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNA(Tyr)-specific probe to Southern blots of Eco RI-digested DNA. A Nicotiana genomic library was prepared by Eco RI digestion of nuclear DNA, ligation of the fragments into the vector lambda gtWES.lambda B and in vitro packaging. The phage library was screened with a 5'-labelled synthetic oligonucleotide complementary to nucleotides 18 to 37 of cytoplasmic tobacco tRNA(Tyr). Eleven hybridizing Eco RI fragments ranging in size from 1.7 to 7.5 kb were isolated from recombinant lambda phage and subcloned into pUC19 plasmid. Four of the sequenced tRNA(Tyr) genes code for the known tobacco tRNA1(Tyr) (G psi A) and seven code for tRNA2(Tyr) (G psi A). The two tRNA species differ in one nucleotide pair at the basis of the T psi C stem. Only one tRNA(Tyr) gene (pNtY5) contains a point mutation (T54-->A54). Comparison of the intervening sequences reveals that they differ considerably in length and sequence. Maturation of intron-containing pre-tRNAs was studied in HeLa and wheat germ extracts. All pre-tRNAs(Tyr)--with one exception--are processed and spliced in both extracts. The tRNA(Tyr) gene encoded by pNtY5 is transcribed efficiently in HeLa extract but processing of the pre-tRNA is impaired.

Base Sequence↗
PubMed1992

Expression of variant nuclear Arabidopsis tRNA(Ser) genes and pre-tRNA maturation differ in HeLa, yeast and wheat germ extracts.

We have recently identified a tRNA gene cluster in the Arabidopsis nuclear genome. One tRNA(Ser) (AGA) gene and two tRNA(Tyr) (GTA) genes occur in tandem arrangement on a 1.5 kb unit that is amplified about 20-fold at a single chromosomal site. Here we have studied the in vitro expression of seven individually cloned tRNA(Ser) genes (pAtS1 to pAtS7) derived from this cluster. Five out of the seven tRNA(Ser) genes contain point mutations in the coding region which have in part adverse effects on the expression of these genes in different cell-free systems: (i) C10 and A62 in plant tRNA(Ser) genes, which correspond to G10 and C62, respectively, in all known vertebrate tRNA genes, result in a reduced transcription efficiency in HeLa but not in yeast extract. This indicates that yeast RNA polymerase III tolerates nucleotide substitutions at positions 10 [5' internal control region (ICR)] and 62 (3' ICR), whereas the vertebrate RNA polymerase III requires a more stringent consensus sequence. (ii) Processing of a pre-tRNA(Ser) with a mismatch in the aminoacyl stem is impaired in HeLa, yeast and wheat germ extracts, however, a mismatch in the anticodon stem is deleterious for HeLa and wheat germ but not for yeast processing enzymes. The unexpectedly high number of potential tRNA(Ser) pseudogenes in the cluster - quite in contrast to the tRNA(Ser) genes which mainly code for functional tRNAs - suggested that tRNA(Ser) (AGA) genes also occur elsewhere in the genome. We present evidence that single copies of tRNA(Ser) (AGA) genes do indeed exist outside the tRNA gene cluster.

Base Sequence↗
PubMed1991

Expression of nuclear tRNA(Tyr) genes from Arabidopsis thaliana in HeLa cell and wheat germ extracts.

The transcription of thirteen Arabidopsis tRNA(Tyr) genes in a tRNA gene cluster was studied in HeLa cell nuclear extracts. All tRNA(Tyr) genes are efficiently transcribed. Initiation of transcription starts at adenosine residues at positions -5 or -6, and termination occurs at an oligo(T) stretch located 17 to 21 bp downstream of the 3' end of the structural genes depending on the gene studied. Maturation of intron-containing pre-tRNAs was compared in HeLa cell and wheat germ extracts and revealed that 5' and 3' end processing precedes splicing in both systems. Two pre-tRNAs which carry mutations in the mature domain are not processed at all in the HeLa extract, whereas one of them matures with low efficiency in the wheat germ extract. The thirteen pre-tRNAs(Tyr) possess highly conserved intron sequences of 12 nucleotides. Twelve of these introns are efficiently excised, whereas one is removed at a reduced rate, possibly due to a slightly destabilized extended anticodon stem.

Base Sequence↗