scholarly journals Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila.

1997 ◽  
Vol 17 (12) ◽  
pp. 7237-7247 ◽  
Author(s):  
P Blomberg ◽  
C Randolph ◽  
C H Yao ◽  
M C Yao
Keyword(s):  
Transcription Initiation ◽  
Tetrahymena Thermophila ◽  
Initiation Site ◽  
Core Region ◽  
Rrna Gene ◽  
Regulatory Sequences ◽  
Coding Region ◽  
Cis Acting ◽  
Rrna Transcription ◽  
Stable Maintenance

We have analyzed the cis-acting sequences that regulate rRNA gene (rDNA) replication in Tetrahymena thermophila. The macronucleus of this ciliated protozoan contains 9,000 copies of a 21-kbp minichromosome in the form of a palindrome comprising two copies of the rDNA. These are derived from a single chromosomally integrated copy during conjugation through selective amplification and are maintained by replicating once per cell cycle during vegetative growth. We have developed a transformation vector and carried out a deletion analysis to determine the minimal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule. Using constructs containing progressively longer deletions, we show that only a small portion (approximately 900 bp) of the rDNA is needed for extrachromosomal replication and stable maintenance of this molecule. This core region is very near but does not include the rRNA transcription initiation site or its putative promoter, indicating that replication is not dependent on normal rRNA transcription. It includes two nearly identical nuclease-sensitive domains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously. Deletion of both domains abolishes replication, whereas deletion of either domain allows the molecules to replicate, indicating that only one domain is required. In addition to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequences within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.

scholarly journals Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin

2008 ◽  
Vol 28 (24) ◽  
pp. 7504-7513 ◽  
Author(s):  
Maxime Tremblay ◽  
Yumin Teng ◽  
Michel Paquette ◽  
Raymond Waters ◽  
Antonio Conconi
Keyword(s):  
Nucleotide Excision Repair ◽  
Transcription Initiation ◽  
Excision Repair ◽  
Initiation Site ◽  
Dna Lesions ◽  
Transcription Initiation Site ◽  
Rrna Gene ◽  
Open Chromatin ◽  
Coding Region ◽  
Nucleotide Excision

ABSTRACT Nucleotide excision repair (NER) removes a plethora of DNA lesions. It is performed by a large multisubunit protein complex that finds and repairs damaged DNA in different chromatin contexts and nuclear domains. The nucleolus is the most transcriptionally active domain, and in yeast, transcription-coupled NER occurs in RNA polymerase I-transcribed genes (rDNA). Here we have analyzed the roles of two members of the xeroderma pigmentosum group C family of proteins, Rad4p and Rad34p, during NER in the active and inactive rDNA. We report that Rad4p is essential for repair in the intergenic spacer, the inactive rDNA coding region, and for strand-specific repair at the transcription initiation site, whereas Rad34p is not. Rad34p is necessary for transcription-coupled NER that starts about 40 nucleotides downstream of the transcription initiation site of the active rDNA, whereas Rad4p is not. Thus, although Rad4p and Rad34p share sequence homology, their roles in NER in the rDNA locus are almost entirely distinct and complementary. These results provide evidences that transcription-coupled NER and global genome NER participate in the removal of UV-induced DNA lesions from the transcribed strand of active rDNA. Furthermore, nonnucleosome rDNA is repaired faster than nucleosome rDNA, indicating that an open chromatin structure facilitates NER in vivo.

scholarly journals Drd4 gene polymorphisms are associated with personality variation in a passerine bird

2007 ◽  
Vol 274 (1619) ◽  
pp. 1685-1691 ◽  
Author(s):  
Andrew E Fidler ◽  
Kees van Oers ◽  
Piet J Drent ◽  
Sylvia Kuhn ◽  
Jakob C Mueller ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Gene Polymorphisms ◽  
Parus Major ◽  
Passerine Bird ◽  
Initiation Site ◽  
Novelty Seeking ◽  
Coding Region ◽  
Free Living ◽  
Single Nucleotide ◽  
Genetic Epistasis

Polymorphisms in several neurotransmitter-associated genes have been associated with variation in human personality traits. Among the more promising of such associations is that between the human dopamine receptor D4 gene ( Drd4 ) variants and novelty-seeking behaviour. However, genetic epistasis, genotype–environment interactions and confounding environmental factors all act to obscure genotype–personality relationships. Such problems can be addressed by measuring personality under standardized conditions and by selection experiments, with both approaches only feasible with non-human animals. Looking for similar Drd4 genotype–personality associations in a free-living bird, the great tit ( Parus major ), we detected 73 polymorphisms (66 SNPs, 7 indels) in the P. major Drd4 orthologue. Two of the P. major Drd4 gene polymorphisms were investigated for evidence of association with novelty-seeking behaviour: a coding region synonymous single nucleotide polymorphism (SNP830) and a 15 bp indel (ID15) located 5′ to the putative transcription initiation site. Frequencies of the three Drd4 SNP830 genotypes, but not the ID15 genotypes, differed significantly between two P. major lines selected over four generations for divergent levels of ‘early exploratory behaviour’ (EEB). Strong corroborating evidence for the significance of this finding comes from the analysis of free-living, unselected birds where we found a significant association between SNP830 genotypes and differing mean EEB levels. These findings suggest that an association between Drd4 gene polymorphisms and animal personality variation predates the divergence of the avian and mammalian lineages. Furthermore, this work heralds the possibility of following microevolutionary changes in frequencies of behaviourally relevant Drd4 polymorphisms within populations where natural selection acts differentially on different personality types.

scholarly journals Expression of the murine alpha B-crystallin gene is not restricted to the lens.

1989 ◽  
Vol 9 (3) ◽  
pp. 1083-1091 ◽  
Author(s):  
R A Dubin ◽  
E F Wawrousek ◽  
J Piatigorsky
Keyword(s):  
Skeletal Muscle ◽  
Transcription Initiation ◽  
Germ Line ◽  
Initiation Site ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
In Vitro System ◽  
Endogenous Gene ◽  
Efficient Expression

The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.

scholarly journals Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca2+-ATPase gene

1995 ◽  
Vol 310 (3) ◽  
pp. 757-763 ◽  
Author(s):  
A Magyar ◽  
E Bakos ◽  
A Váradi
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Drosophila Genome ◽  
Coding Region ◽  
Specific Expression ◽  
S1 Nuclease ◽  
Protein Coding ◽  
Atpase Gene ◽  
High Level

A 14 kb genomic clone covering the organellar-type Ca(2+)-ATPase gene of Drosophila melanogaster has been isolated and characterized. The sequence of a 7132 bp region extending from 1.1 kb 5′ upstream of the initiation ATG codon over the polyadenylation signal at the 3′ end has been determined. The gene consists of nine exons including one with an exceptional size of 2172 bp representing 72% of the protein coding region. Introns are relatively small (< 100 bp) except for the 3′ intron which has a size of 2239 bp, an exceptionally large size among Drosophila introns. Five of the introns are in the same positions in Drosophila, Artemia and rabbit SERCA1 Ca(2+)-ATPase genes. There is only one organellar-type Ca(2+)-ATPase gene in the Drosophila genome, as was shown by Southern-blot analysis [Váradi, Gilmore-Hebert and Benz (1989) FEBS Lett. 258, 203-207] and by chromosomal localization [Magyar and Váradi (1990) Biochem. Biophys. Res. Commun. 173, 872-877]. Primer extension and S1-nuclease assays revealed a potential transcription initiation site 876 bp upstream of the translation initiation ATG with a TATA-box 23 bp upstream of this site. Analysis of the 5′ region of the Drosophila organellar-type Ca(2+)-ATPase gene suggests the presence of potential recognition sequences of various muscle-specific transcription factors and shows a region with remarkable similarity to that in the rabbit SERCA2 gene. The tissue distribution of expression of the organellar-type Ca(2+)-ATPase gene has been studied by in situ RNA-RNA hybridization on microscopic sections. A low mRNA abundance can be detected in each tissue of adult flies, suggesting a housekeeping function for the gene. On the other hand a pronounced tissue specificity of expression has also been found as the organellar-type Ca(2+)-ATPase is expressed at a very high level in cell bodies of the central nervous system and in various muscles.

scholarly journals The Genes Encoding Formamidopyrimidine and MutY DNA Glycosylases in Escherichia coli Are Transcribed as Part of Complex Operons

1999 ◽  
Vol 181 (14) ◽  
pp. 4223-4236 ◽  
Author(s):  
Christine M. Gifford ◽  
Susan S. Wallace
Keyword(s):  
Escherichia Coli ◽  
Gene Order ◽  
Intergenic Region ◽  
Transcription Initiation ◽  
Excision Repair ◽  
Initiation Site ◽  
Dna Glycosylase ◽  
Rnase E ◽  
Coding Region ◽  
Genes Encoding

ABSTRACT Escherichia coli formamidopyrimidine (Fpg) DNA glycosylase and MutY DNA glycosylase are base excision repair proteins that work together to protect cells from the mutagenic effects of the commonly oxidized guanine product 7,8-dihydro-8-oxoguanine. The genes encoding these proteins, fpg and mutY, are both cotranscribed as part of complex operons. fpg is the terminal gene in an operon with the gene order radC,rpmB, rpmG, and fpg. This operon has transcription initiation sites upstream of radC, in theradC coding region, and immediately upstream offpg. There is a strong attenuator in therpmG-fpg intergenic region and three transcription termination sites downstream of fpg. There is an additional site, in the radC-rpmB intergenic region, that corresponds either to a transcription initiation site or to an RNase E or RNase III cleavage site. mutY is the first gene in an operon with the gene order mutY, yggX, mltC, andnupG. This operon has transcription initiation sites upstream of mutY, in the mutY coding region, and immediately upstream of nupG. There also appear to be attenuators in the yggX-mltC and mltC-nupGintergenic regions. The order of genes in these operons has been conserved or partially conserved only in other closely related gram-negative bacteria, although it is not known whether the genes are cotranscribed in these other organisms.

scholarly journals Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region.

1989 ◽  
Vol 9 (8) ◽  
pp. 3244-3252 ◽  
Author(s):  
J M Huibregtse ◽  
D R Engelke
Keyword(s):  
Stationary Phase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Dnase I ◽  
Transcription Initiation Site ◽  
Upstream Region ◽  
Stable Complex ◽  
Coding Region ◽  
Flanking Region ◽  
Genomic Footprinting

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.

scholarly journals Genetic evidence for promoter competition in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4608-4615 ◽  
Author(s):  
J E Hirschman ◽  
K J Durbin ◽  
F Winston
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Base Change ◽  
Wild Type ◽  
Base Pairs ◽  
Cis Acting ◽  
Initiation Of Transcription ◽  
In Trans ◽  
Tata Sequence ◽  
Transcription Signals

The his4-912 delta mutation is an insertion of the long terminal repeat (delta) of the yeast retrotransposon Ty into the HIS4 promoter region, such that the delta is 97 base pairs upstream of the HIS4 transcription initiation site. Strains carrying the his4-912 delta allele are His- at 23 degrees C; this phenotype can be reversed either by growth at 37 degrees C or by mutations in trans-acting SPT genes. Under conditions in which his4-912 delta confers a His- phenotype. HIS4 transcription initiates at the delta initiation site, rather than at the HIS4 initiation site, producing a longer, nonfunctional transcript. Under conditions in which the strain is His+, transcription initiates at the wild-type HIS4 initiation site. To understand how transcription is balanced between the delta and HIS4 promoters, we have selected for cis-acting suppressors of his4-912 delta. Two classes defined by six independent mutations restore synthesis of a functional HIS4 transcript. The first class is an A-to-G base change 1 base upstream of the proposed delta TATA sequence. These mutants do not synthesize the delta-initiated transcript; instead, they synthesize only the wild-type HIS4 transcript. The second class of mutations alters base pairs surrounding the functional HIS4 TATA sequence. The two strongest His+ mutants of this class synthesize the wild-type HIS4 transcript at levels consistent with their His+ phenotype. Surprisingly, these two mutants also have a reduced level of the delta-initiated transcript relative to the his4-912 delta parent. Analysis of these mutants indicates that the level of transcription from one promoter can affect the level of transcription from the other promoter and suggests that delta and HIS4 transcription signals compete for initiation of transcription from each site.

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