Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity

Methylmalonyl-CoA mutase (MCM) is a nuclear-encoded mitochondrial matrix enzyme. We have reported characterization of murine MCM and cloning of a murine MCM cDNA and now describe the murine Mut locus, its promoter and evidence for tissue-specific variation in MCM mRNA, enzyme and holo-enzyme levels. The Mut locus spans 30 kb and contains 13 exons constituting a unique transcription unit. A B1 repeat element was found in the 3′ untranslated region (exon 13). The transcription initiation site was identified and upstream sequences were shown to direct expression of a reporter gene in cultured cells. The promoter contains sequence motifs characteristic of: (1) TATA-less housekeeping promoters; (2) enhancer elements purportedly involved in co-ordinating expression of nuclear-encoded mitochondrial proteins; and (3) regulatory elements including CCAAT boxes, cyclic AMP-response elements and potential AP-2-binding sites. Northern blots demonstrate a greater than 10-fold variation in steady-state mRNA levels, which correlate with tissue levels of enzyme activity. However, the ratio of holoenzyme to total enzyme varies among different tissues, and there is no correlation between steady-state mRNA levels and holoenzyme activity. These results suggest that, although there may be regulation of MCM activity at the level of mRNA, the significance of genetic regulation is unclear owning to the presence of epigenetic regulation of holoenzyme formation.

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Genotype-dependent and non-gradient patterns of RSV gene expression

10.1101/641878 ◽

2019 ◽

Author(s):

Felipe-Andrés Piedra ◽

Xueting Qiu ◽

Michael N. Teng ◽

Vasanthi Avadhanula ◽

Annette A. Machado ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Steady State ◽

Transcription Initiation ◽

Mrna Levels ◽

Viral Pathogen ◽

Negative Strand ◽

Gene Start ◽

Syncytial Virus ◽

Conserved Gene

AbstractRespiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA virus and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3’ promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription. However, recent reports show data that appear inconsistent with a gradient. To better understand RSV transcriptional regulation, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and cotton rats infected with virus isolates belonging to four different genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were genotype-specific and non-gradient, where mRNA levels from the G (attachment) gene exceeded those from the more promoter-proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. While the GS signal sequences were highly conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, was variable, suggesting polymerase recognition of GS signal conformation affects transcription initiation. The effect of GS N-phase on transcription efficiency was tested using dicistronic minigenomes. Ratios of minigenome gene expression showed a switch-like dependence on N-phase with a period of seven nucleotides. Our results indicate that RSV gene expression is in part sculpted by polymerases that initiate transcription with a probability dependent on GS signal N-phase.Author SummaryRSV is a major viral pathogen that causes significant morbidity and mortality, especially in young children. Shortly after RSV enters a host cell, transcription from its nonsegmented negative-strand (NNS) RNA genome starts at the 3’ promoter and proceeds sequentially. Transcriptional attenuation is thought to occur at each gene junction, resulting in a gradient of gene expression. However, recent studies showing non-gradient levels of RSV mRNA suggest that transcriptional regulation may have additional mechanisms. We show using RSV isolates belonging to four different genotypes that gene expression is genotype-dependent and one gene (the G or attachment gene) is consistently more highly expressed than an upstream neighbor. We hypothesize that variable alignment of highly conserved gene start (GS) signals with nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-gradient patterns of gene expression. We show using dicistronic RSV minigenomes wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase affects gene expression. Our results suggest the existence of a novel mechanism of transcriptional regulation that might play a role in other NNS RNA viruses.

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Aqp1 expression in erythroleukemia cells: genetic regulation of glucocorticoid and chemical induction

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.5.c1562 ◽

1997 ◽

Vol 273 (5) ◽

pp. C1562-C1570 ◽

Cited By ~ 78

Author(s):

Chulso Moon ◽

Landon S. King ◽

Peter Agre

Keyword(s):

Transcriptional Regulation ◽

Transcription Initiation ◽

Protein Biosynthesis ◽

Genetic Regulation ◽

Water Channel ◽

Erythroid Differentiation ◽

Regulatory Elements ◽

Proximal Promoter ◽

Chemical Induction ◽

Site Mutation

The aquaporin-1 (AQP1) water channel protein is expressed in multiple mammalian tissues by several different developmental programs; however, the genetic regulation is undefined. The proximal promoter of mouse Aqp1 contains multiple putative cis-acting regulatory elements, and mouse erythroleukemia (MEL) cells are a well-characterized model for erythroid differentiation. Corticosteroid or dimethyl sulfoxide (DMSO) exposure induces AQP1 protein expression in MEL cells, and transcriptional regulation was investigated by transient transfections with Aqp1 promoter-reporter constructs. Dexamethasone induction is abrogated by deletion of two glucocorticoid response elements −0.5 kilobases (kb) from the transcription initiation site. Mutation of the GATA element at −0.62 kb has no effect, whereas mutation of the CACCC site at −37 bp significantly reduces DMSO-induced promoter activity. Hydroxyurea induces expression of AQP1 protein without acting through the proximal promoter. The MEL cell line is a reproducible erythroid model system for studying transcriptional regulation of the Aqp1 gene while determining the consequences on AQP1 protein biosynthesis.

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Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6191 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6191-6200 ◽

Cited By ~ 59

Author(s):

Yukako Yamabe ◽

Akira Shimamoto ◽

Makoto Goto ◽

Jun Yokota ◽

Minoru Sugawara ◽

...

Keyword(s):

Transcription Initiation ◽

Promoter Activity ◽

Transcriptional Control ◽

Housekeeping Genes ◽

Regulatory Elements ◽

Initiation Site ◽

Luciferase Reporter ◽

Upstream Region ◽

Control Element ◽

Mrna Levels

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

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Molecular structure and organization of the wheat genomic manganese superoxide dismutase gene

Genome ◽

10.1139/g05-102 ◽

2006 ◽

Vol 49 (3) ◽

pp. 209-218 ◽

Cited By ~ 16

Author(s):

Kwang-Hyun Baek ◽

Daniel Z Skinner ◽

Peng Ling ◽

Xianming Chen

Keyword(s):

Superoxide Dismutase ◽

Transposable Element ◽

Transcription Initiation ◽

Manganese Superoxide Dismutase ◽

Genomic Structure ◽

Regulatory Elements ◽

Manganese Superoxide ◽

Superoxide Dismutase Gene ◽

Responsive Element ◽

Mnsod Gene

The genomic structure of a manganese superoxide dismutase (MnSOD) gene in wheat was elucidated by sequencing a clone from a BAC library of a stripe rust resistant wheat line. The clone was identified by hybridization with a wheat MnSOD cDNA. The gene consisted of 6 exons interrupted by 5 introns with a total length of 4770 nucleotides from the start codon to the termination codon. The wheat MnSOD gene was the longest among those sequenced from plant species. The transcription initiation site was preceded by a G+C-rich promoter without a TATA or CAAT box. The promoter contained many putative cis-acting regulatory elements, including an abscisic acid (ABA)-responsive element, a stress-responsive element, and a GC-repeat, as well as several other structural features in common with the promoter of the rice MnSOD gene. A Stowaway-like transposable element was found in intron 5 of the wheat MnSOD gene, but further investigation revealed the transposable element was not present in all copies of the MnSOD genes.Key words: AY963808, BAC clone, manganese superoxide dismutase, gene, promoter, transposable element.

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TRAP-based allelic translation efficiency imbalance analysis to identify genetic regulation of ribosome occupancy in specific cell types in vivo

10.1101/2020.08.24.265389 ◽

2020 ◽

Author(s):

Yating Liu ◽

Anthony D. Fischer ◽

Celine L. St. Pierre ◽

Juan F. Macias-Velasco ◽

Heather A. Lawson ◽

...

Keyword(s):

Gene Expression ◽

Genetic Regulation ◽

Transcript Abundance ◽

Cell Types ◽

Regulatory Elements ◽

Model Organisms ◽

Specific Cell ◽

Translation Efficiency ◽

Mrna Levels

AbstractThe alteration of gene expression due to variations in the sequences of transcriptional regulatory elements has been a focus of substantial inquiry in humans and model organisms. However, less is known about the extent to which natural variation contributes to post-transcriptional regulation. Allelic Expression Imbalance (AEI) is a classical approach for studying the association of specific haplotypes with relative changes in transcript abundance. Here, we piloted a new TRAP based approach to associate genetic variation with transcript occupancy on ribosomes in specific cell types, to determine if it will allow examination of Allelic Translation Imbalance (ATI), and Allelic Translation Efficiency Imbalance, using as a test case mouse astrocytes in vivo. We show that most changes of the mRNA levels on ribosomes were reflected in transcript abundance, though ∼1.5% of transcripts have variants that clearly alter loading onto ribosomes orthogonally to transcript levels. These variants were often in conserved residues and altered sequences known to regulate translation such as upstream ORFs, PolyA sites, and predicted miRNA binding sites. Such variants were also common in transcripts showing altered abundance, suggesting some genetic regulation of gene expression may function through post-transcriptional mechanisms. Overall, our work shows that naturally occurring genetic variants can impact ribosome occupancy in astrocytes in vivo and suggests that mechanisms may also play a role in genetic contributions to disease.

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Co-ordinate expression of cytochrome c oxidase subunit III and VIc mRNAs in rat tissues

Biochemical Journal ◽

10.1042/bj2690503 ◽

1990 ◽

Vol 269 (2) ◽

pp. 503-506 ◽

Cited By ~ 40

Author(s):

D A Hood

Keyword(s):

Enzyme Activity ◽

Steady State ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Mrna Level ◽

Rat Tissues ◽

Mrna Levels ◽

Subunit Mrna ◽

Oxidase Subunit ◽

Subunit Iii

Cytochrome c oxidase (EC 1.9.3.1) is an enzyme which is composed of subunits derived from both the mitochondrial and the nuclear genomes. To determine whether or not the expression of these two genomes is co-ordinated at the mRNA level, we have examined the steady-state levels of mRNAs coding for cytochrome c oxidase subunit III (mitochondrially encoded) and subunit VIc (nuclear-encoded) in rat tissues. This was compared with the tissue concentration of the holoenzyme, which was estimated by measuring cytochrome c oxidase enzyme activity. The tissues (heart, brain, liver, kidney, soleus muscle and superficial white vastus muscle) possessed a 13-fold range of enzyme activity, which was highest in heart and lowest in the superficial vastus muscle. Specific subunit mRNA levels were quantified by using slot-blot hybridization of cDNA probes to total tissue RNA. The highest values for subunit III and Vlc mRNA tissue contents were found in kidney, followed by liver and heart (40-60% of that of kidney). The white vastus muscle contained the lowest subunit mRNA level (15% of that of kidney). Although some variability was apparent within each tissue, a parallel pattern of mRNA expression of the nuclear- and mitochondrially encoded subunits was observed. Differences between muscle (heart, vastus and soleus) and non-muscle tissues were noted in the relationship between mRNA and protein levels of expression. Thus, although this suggests that tissue-specific regulatory processes operate, the steady-state expression of subunit III and subunit Vlc mRNAs appears to be co-ordinately regulated.

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Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients

Development ◽

10.1242/dev.126.11.2527 ◽

1999 ◽

Vol 126 (11) ◽

pp. 2527-2538 ◽

Cited By ~ 13

Author(s):

M. Fujioka ◽

Y. Emi-Sarker ◽

G.L. Yusibova ◽

T. Goto ◽

J.B. Jaynes

Keyword(s):

Regulatory Elements ◽

Transcription Unit ◽

Single Element ◽

Mrna Levels ◽

Protein Levels ◽

Cis Element ◽

Gap Gene ◽

Mother Cells ◽

Regulatory Dna ◽

Blastoderm Stage

The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3′ of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Kruppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is ‘carved out’ by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314–24), but at different concentrations. The 3′ region also contains a single element for neurogenic expression in ganglion mother cells 4–2a and 1–1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3′ untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3′ end of the locus, but was not found to contribute to efficient functional rescue.

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Multiple Pathways for Steel Regulation Suggested by Genomic and Sequence Analysis of the Murine Steel Gene

Genetics ◽

10.1093/genetics/142.3.927 ◽

1996 ◽

Vol 142 (3) ◽

pp. 927-934 ◽

Cited By ~ 2

Author(s):

Mary A Bedell ◽

Neal G Copeland ◽

Nancy A Jenkins

Keyword(s):

Binding Sites ◽

Transcription Initiation ◽

Expression Patterns ◽

Initiation Site ◽

Transcription Unit ◽

Full Length ◽

Sequence Motifs ◽

Mouse Tissues ◽

Polyadenylation Sites ◽

Flanking Region

Abstract The Steel (Sl) locus encodes mast cell growth factor (Mgf) that is required for the development of germ cells, hematopoietic cells and melanocytes. Although the expression patterns of the Mgf gene are well characterized, little is known of the factors which regulate its expression. Here, we describe the cloning and sequence of the full-length transcription unit and the 5′ flanking region of the murine Mgf gene. The full-length Mgf mRNA consists of a short 5′ untranslated region (UTR), a 0.8-kb ORF and a long 3′ UTR. A single transcription initiation site is used in a number of mouse tissues and is located just downstream of binding sites for several known transcription factors. In the 5′ UTR, two ATGs were found upstream of the initiator methionine and are conserved among different species, suggesting that Mgf may be translationally regulated. At least two Mgf mRNAs are produced by alternative use of polyadenylation sites, but numerous other potential polyadenylation sites were found in the 3′ UTR. In addition, the 3′ UTR contains numerous sequence motifs that may regulate Mgf mRNA stability. These studies suggest multiple ways in which expression of Mgf may be regulated.

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Transcriptional Analysis of the Conserved ftsZ Gene Cluster in Mycoplasma genitalium and Mycoplasma pneumoniae

Journal of Bacteriology ◽

10.1128/jb.187.13.4542-4551.2005 ◽

2005 ◽

Vol 187 (13) ◽

pp. 4542-4551 ◽

Cited By ~ 18

Author(s):

Gwynedd A. Benders ◽

Bradford C. Powell ◽

Clyde A. Hutchison

Keyword(s):

Gene Cluster ◽

Mycoplasma Pneumoniae ◽

Transcription Initiation ◽

Mycoplasma Genitalium ◽

Transcription Unit ◽

Transcriptional Analysis ◽

Pcr Analysis ◽

Mrna Levels ◽

Initiation Point ◽

Six Genes

ABSTRACT Several experimental approaches were used to construct a detailed transcriptional profile of the phylogenetically conserved ftsZ cell division gene cluster in both Mycoplasma genitalium and its closest relative, Mycoplasma pneumoniae. We determined initiation and termination points for the cluster, as well as an absolute steady-state RNA level for each gene. Transcription of this cluster in both these organisms was shown to be highly strand specific. While the four genes in this cluster are cotranscribed, their transcription unit also includes two genes of close proximity yet disparate function. A transcription initiation point immediately upstream of these two genes was detected in M. genitalium but not M. pneumoniae. In M. pneumoniae, transcription of the six genes terminates at a poly(U)-tailed hairpin. In M. genitalium, this transcription terminates at two closely spaced points by an unknown mechanism. Real-time reverse transcription-PCR analysis of this cluster in M. pneumoniae shows that mRNA levels for all six genes vary at most fivefold and form a gradient of decreasing quantity with increasing distance from the promoter at the beginning of the cluster. mRNA from coding regions was approximately 20- to 100-fold more abundant than that from intergenic regions. We estimated the most abundant mRNA we detected at 0.6 copy per cell. We conclude that groups of functionally related genes in M. genitalium and M. pneumoniae are often preceded by promoters but rarely followed by terminators. This causes functionally unrelated genes to be commonly cotranscribed in these organisms.

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Structure of the 5′ region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites

Biochemical Journal ◽

10.1042/bj3590129 ◽

2001 ◽

Vol 359 (1) ◽

pp. 129-137 ◽

Cited By ~ 3

Author(s):

Dorota ŚCIEGLIŃSKA ◽

Wiesława WID̄ŁAK ◽

Witold KONOPKA ◽

Matti POUTANEN ◽

Nafis RAHMAN ◽

...

Keyword(s):

Transcription Initiation ◽

Gene Promoter ◽

Regulatory Elements ◽

Transcription Unit ◽

Northern Blotting ◽

Start Codon ◽

Specific Expression ◽

Rnase Protection ◽

Exon 1 ◽

Testis Specific Expression

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5′ region of the transcription unit of these genes as well as localization of the ‘cis’ elements conferring their testis-specific expression gave contradictory results [Widłak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191–200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310–321]. In the present paper we solve these controversies and show that the 5′ untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5′ UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on ‘cis’ regulatory elements localized within exon 1 and the intron sequences.

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