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PubMed2006

Mitochondrial transcription is regulated via an ATP "sensing" mechanism that couples RNA abundance to respiration.

The information encoded in both the nuclear and mitochondrial genomes must be coordinately regulated to respond to changes in cellular growth and energy states. Despite identification of the mitochondrial RNA polymerase (mtRNAP) from several organisms, little is known about mitochondrial transcriptional regulation. Studying the shift from fermentation to respiration in Saccharomyces cerevisiae, we have demonstrated a direct correlation between in vivo changes in mitochondrial transcript abundance and in vitro sensitivity of mitochondrial promoters to ATP concentration (K(m)ATP). Consistent with the idea that the mtRNAP itself senses in vivo ATP levels, we found that transcript abundance correlates with respiration, but only when coupled to mitochondrial ATP synthesis. In addition, we characterized mutations in the mitochondrial promoter and the mtRNAP accessory factor Mtf1 that alter both in vitro K(m)ATP and in vivo transcription in response to respiratory changes. We propose that shifting cellular pools of ATP coordinately control nuclear and mitochondrial transcription.

Adenosine Triphosphate↗
PubMed1990

Characterization of human parainfluenza virus type 3 persistent infection in cell culture.

Three cell lines persistently infected with human parainfluenza virus type 3 were characterized on a molecular level in this study. All six structural protein genes were transcribed into monocistronic RNAs in the persistently infected cells. In both acutely and persistently infected cells, polycistronic transcripts were abundant, although the ratio of polycistronic to monocistronic transcripts was reduced in the persistently infected cells. Each of the persistently infected cell lines contained a distinct subgenomic RNA species. The subgenomic RNAs were present in purified nucleocapsid cores, indicating that they represent viral genome RNA, were far more abundant than full-length RNA, and were stably maintained through at least 36 cell passages. Nucleotide sequence analysis of the subgenomic RNAs from two of the persistently infected cell lines revealed that the 5' ends are identical to that of the standard genome. Hybridization experiments with oligonucleotide probes showed that both fragments retain sequences from the 5' end of the standard genome and contain approximately 1,200 nucleotides (cell line 1) and 1,500 nucleotides (cell line 2) of the polymerase gene sequence. The demonstration of several alterations in viral gene expression in persistently infected cells offers insight into the factors associated with persistence of parainfluenza virus 3.

Animals↗
PubMed1995

Differential expression of two 1-aminocyclopropane-1-carboxylic acid oxidase genes in broccoli after harvest.

Broccoli (Brassica oleracea L.) floral tissues rapidly differentiate and grow before harvest and then senesce rapidly after harvest. Associated with this postharvest deterioration is an increase in ethylene production by florets. Two cDNA clones having high nucleotide identity to sequences encoding 1-amino-cyclopropane-1-carboxylic acid (ACC) oxidase were isolated from senescing florets. The cDNAs, ACC Ox1 and ACC Ox2, apparently encode mRNAs from different genes. ACC Ox1 transcripts were found at low levels in whole florets at the time of harvest and increased markedly in abundance after harvest. ACC Ox1 transcript abundance also increased in sepals after harvest and in excised yellowing leaves. Transcripts corresponding to ACC Ox2 were found exclusively within the reproductive structures. These ACC Ox2 transcripts were absent at harvest but started to increase in abundance within 2 h of harvest and then accumulated to high levels. Hormone treatment did not alter the abundance of ACC Ox1 transcripts, whereas ACC Ox2 transcripts increased in abundance after treatment with abscisic acid and propylene. Wounding did not affect the levels of ACC Ox1 or Ox2 transcripts after harvest. At harvest, individual broccoli florets were closed and remained unpollinated. We propose a model whereby the rapid increase in ACC Ox1 and Ox2 transcript abundance after harvest contributes to increased ethylene production by florets. This ethylene may regulate aspects of postharvest senescence, in particular chlorophyll loss.

Amino Acid Oxidoreductases↗
PubMed1998

Human sex hormone-binding globulin gene expression in transgenic mice.

The sex hormone-binding globulin gene (shbg) is expressed in the liver and testis as well as in several other tissues that play important roles in reproduction. Expression of shbg in the human liver results in the production of plasma sex hormone-binding globulin (SHBG), which regulates the bioavailability of sex steroids in the blood. Although shbg is not expressed in rodent livers postnatally, it gives rise to the androgen-binding protein in their testes upon sexual maturation. Human shbg is also expressed in the testis, but its products and their function are less well characterized. To study the expression of human shbg in different tissues and the consequences of overexpressing this gene in vivo, we have produced several lines of mice containing approximately 11-kilobase (kb; shbg11) or 4.3-kb (shbg4) human shbg genomic fragments that comprise all eight exons encoding SHBG as well as approximately 6 kb or approximately 0.9 kb of 5'-flanking DNA, respectively. Northern blots indicated that human shbg transcripts were most abundant in liver, kidney, and testis of the shbg11 mice. The 4.3-kb shbg transgenes were expressed at similar levels in liver and kidney, but the abundance of human shbg transcripts in their testes was much lower than that in shbg11 mice. Primer extension analysis indicated that transcription starts 60 bp from the translation initiation codon for SHBG in liver and kidney of shbg11 mice, and that the shbg transcripts in their testis are derived from a separate promoter flanking an alternative exon that replaces the exon containing the translation initiation codon for SHBG or androgen-binding protein. At the cellular level, the human shbg transgenes are expressed in clusters of hepatocytes located mainly within the periportal region of hepatic lobules and in the epithelial cells lining the proximal convoluted tubules of the kidney. This results in high levels of human SHBG in serum (1.45-1.72 nmol/ml) and urine (6-16 pmol/ml) of mature male shbg mice. The abundance and distribution of human shbg transcripts in the Sertoli cells of shbg11 mice vary throughout the spermatogenic cycle, with levels increasing in the Sertoli cell cytoplasm until stage VII of spermatogenesis and declining after stage IX. At stages X-XII of spermatogenesis, these transcripts concentrate at the adluminal compartment of the Sertoli cells, and this suggests that they have a role in the elongation phase of spermiogenesis. The presence of human SHBG in the blood of shbg transgenic mice may result in serum levels of testosterone that are 10-100 times higher than those in wild-type littermates. Despite this, their reproductive performance is normal, and there is no obvious phenotypic abnormalities even in animals homozygous for the transgenes.

Animals↗
PubMed2004

Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR.

ACAT (also called sterol o-acyltransferase) catalyzes the esterification of cholesterol by reaction with long-chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to beta-actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n = 17), ACAT-1 transcripts were on average 9-fold (range, 1.7- to 167-fold) more abundant than ACAT-2, whereas in duodenal samples (n = 10), ACAT-2 transcripts were on average 3-fold (range, 0.39- to 12.2-fold) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine.

Duodenum↗
PubMed1994

Retinoic acid and mouse skin morphogenesis. I. Expression pattern of retinoic acid receptor genes during hair vibrissa follicle, plantar, and nasal gland development.

The spatial and temporal expression of the nuclear retinoic acid receptors alpha, beta, and gamma (RAR-alpha, beta, and gamma) was compared by in situ hybridization during hair vibrissa follicle and nasal and plantar eccrine gland morphogenesis in mouse embryo. The RAR-alpha and RAR-gamma transcripts are abundant in the dermal papilla cells of the hair vibrissa when these cells elicit epidermal hair placode (12.5-d embryos) and hair follicle (13.5-d embryos) formation. Both these transcripts are also abundant in the dermal cells of the plantar foot pad at the initiation stage (17.5-d embryos) of glandular morphogenesis. In epidermal cells, the distribution of RAR-gamma transcripts increases in parallel with hair vibrissa follicle and sweat gland differentiation, and thus may be part of the epidermal response to the dermal instructions. The RAR-beta signal is barely above control level during both hair vibrissa and plantar gland morphogenesis. By contrast, during nasal gland formation (12.5- to 15.5-d embryos), the RAR-beta signal reaches a high level in mesenchymal cells, whereas the RAR-alpha-transcripts are present in both epithelial and mesenchymal cells. These results suggest a role for RAR-alpha and RAR-gamma in the epidermal-dermal interactions that lead to hair follicle and plantar gland morphogenesis, whereas the nasal gland development implies RAR-alpha and RAR-beta gene expression. This should be correlated with the expression of the RAR-beta gene that was previously shown to be linked to the RA-induced glandular metaplasia of hair vibrissa follicles.

Animals↗
PubMed1987

Cloning and sequence of rat myoadenylate deaminase cDNA. Evidence for tissue-specific and developmental regulation.

Myoadenylate deaminase is the muscle-specific isoform of AMP deaminase (EC 3.5.4.6), an enzyme which plays a special role in energy metabolism in skeletal muscle. A 2.3-kilobase cDNA encoding this enzyme has been cloned from a lambda gt10 library prepared from rat skeletal muscle using oligonucleotide probes designed from AMP deaminase peptide sequences. This cDNA was sequenced, and the amino acid sequence of this isoform of AMP deaminase was deduced. Sequences homologous to this cDNA are identified in the genome of eukaryotes as diverse as yeast and man. Tissue-specific expression of a 2.5-kilobase AMP deaminase transcript is demonstrated, and the abundance of this transcript as well as the 80-kDa adult, muscle-specific peptide of AMP deaminase increase in parallel during postnatal skeletal muscle development. In the adult animal, the abundance of this transcript and AMP deaminase activity are differentially expressed in various skeletal muscle fiber types. We conclude that AMP deaminase sequences have been highly conserved during evolution, and in mammals there is developmental and tissue-specific control of expression of this gene.

AMP Deaminase↗
PubMed2004

Changes in the endoplasmic reticulum lipid properties in response to low temperature in Brassica napus.

Cold is an abiotic stress known to induce changes in membrane lipid composition. However, there is only limited information on the differential reactivity to environmental temperature of distinct cellular compartments. Therefore, we focused our attention on the endoplasmic reticulum (ER) that was never studied in this respect in plants. The ER membranes of etiolated Brassica napus (oilseed rape) hypocotyls grown at low temperature (4 degrees C) has been shown to be enriched in polyunsaturated fatty acids and phosphatidylethanolamine (PtdEtn) compared to hypocotyls grown at 22 degrees C. Despite the significant changes in their lipid composition upon cold exposure, the ER membranes showed a very partial physico-chemical adaptation as determined by measurement of membrane fluidity parameters such as local microviscosity of acyl chains and lipid lateral diffusion. To investigate the implication of transcriptional regulations during cold acclimation, we compared the abundance of transcripts for genes related to the fatty acid and the phosphatidylcholine (PtdCho)/PtdEtn biosynthesis pathways between normal temperature (22 degrees C)-acclimated and cold temperature (4 degrees C)-treated seedlings, using heterologous cDNA-array technology based on the knowledge on the Arabidopsis genome. Our studies demonstrate that a putative stearoyl-ACP desaturase isogene (orthologous to At1g43800) was up-regulated in response to low temperature.

Adaptation, Physiological↗
PubMed2000

cDNA cloning and expression analysis of a non-photosynthetic ferredoxin gene in morning glory (Pharbitis nil).

A full-length cDNA encoding a non-photosynthetic ferredoxin was isolated from apical buds of morning glory (Pharbitis nil), a short-day plant, by differential screening under flower-inducing and non-inducing conditions. Northern analysis and in situ hybridization showed that the transcript was abundant in shoot apices and root tips. The transcript level in the apical buds decreased with the flower-inducing light treatment.

Amino Acid Sequence↗
PubMed2002

Na,K-ATPase alpha and beta subunit genes exhibit unique expression patterns during zebrafish embryogenesis.

We have used in situ hybridization to analyze Na,K-ATPase alpha and beta subunit gene expression during zebrafish embryogenesis. The most striking finding is that each of the 14 Na,K-ATPase genes exhibits a distinct expression profile. All alpha and beta subunit genes are expressed in the nervous system, although the pattern of expression in different regions varies dramatically. In peripheral tissues, three of the five alpha1-like genes are expressed in pronephros and mucous cells, one is expressed in heart, and one is predominant in skeletal muscle. The alpha2 gene is expressed in brain and heart but is most prominent in skeletal muscle, while the two alpha3 genes are restricted in their expression to the nervous system. Of the six beta subunit genes, beta1a is expressed at highest abundance in lens, pronephros, and heart, while beta1b transcripts are abundant in mucous cells. The two beta2-like genes are differentially expressed in the nervous system. One beta3 gene is expressed exclusively in brain while the other is abundantly expressed in skeletal muscle. Based on these expression patterns, we predict that at least 14 alpha/beta subunit pairs are likely to be formed in different tissues.

Animals↗
PubMed1995

A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA: location of a cryptic CYP21 promoter element?

Complement component C4 is encoded by two nearly identical genes, C4A and C4B, that encode a C4 precursor that is proteolytically cleaved into the alpha, beta and gamma subunits of the mature protein. C4 is expressed primarily in liver and to a much lesser extent in immune cells. We have identified a unique 1 kb RNA transcript, termed Z, that arises from a cryptic promoter lying in the intron between exons 35 and 36 of the C4 gene. Primer extension, RNase protection, and 5' RACE experiments locate the cap site in intron 35, 55 bases upstream from exon 36. Northern blotting and RNase protection assays show that expression of this 1 kb Z RNA transcript is confined to the adrenal gland. Z RNA contains the same open reading frame as C4 which predicts a protein of 131 amino acids, but antisera to C4 do not interact with epitopes on this protein when it is synthesized by cell-free translation, hence the presence or absence of a Z protein in vivo could not be determined. Transfection of Z promoter/reporter constructs into human adrenal NCI-H295 cells shows that most if not all of the sequences required for high-level adrenal expression lie within 235 bases upstream from the cap site, but that this region is inactive when transfected into COS-1, JEG-3 and Hep-G2 cells, suggesting it contains an adrenal-specific element. The 222 bases upstream from the cap site are 75% identical in the human C4A and mouse Slp genes, and contain a potential binding site for steroidogenic factor 1 (SF-1), an orphan zinc-finger nuclear receptor. We propose that this region, like a nearby region in the mouse genome, functions as an upstream element of the P450c21 promoter, and may be a component of an adrenal-specific locus-control region.

Adrenal Glands↗
PubMed2001

Expression of stimulator of Fe transport is not enhanced in Hfe knockout mice.

Hfe knockout (-/-) mice recapitulate many of the biochemical abnormalities of hereditary hemochromatosis (HH), but the molecular mechanisms involved in the etiology of iron overload in HH remain poorly understood. It was found previously that livers of patients with HH contained 5-fold higher SFT (stimulator of Fe transport) mRNA levels relative to subjects without HH. Because this observation suggests a possible role for SFT in HH, we investigated SFT mRNA expression in Hfe(-/-) mice. The 4- and 10-wk-old Hfe(-/-) mice do not have elevated levels of hepatic SFT transcripts relative to age-matched Hfe(+/+) mice, despite having 2.2- and 3.3-fold greater hepatic nonheme iron concentrations, respectively. Northern blot analyses of various mouse tissues revealed that SFT is widely expressed. The novel observation that SFT transcripts are abundant in brain prompted a comparison of SFT transcript levels and nonheme iron levels in the brains of Hfe(+/+) and Hfe(-/-) mice. Neither SFT mRNA levels nor nonheme iron levels differed between groups. Further comparisons of Hfe(-/-) and Hfe(+/+) mouse tissues revealed no significant differences in SFT mRNA levels in duodenum, the site of increased iron absorption in HH. Important distinctions between Hfe(-/-) mice and HH patients include not only differences in the relative rate and magnitude of iron loading but also the lack of fibrosis and phlebotomy treatment in the knockout animals.

Animals↗
PubMed1993

kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus.

The kil-kor regulon of IncP plasmid RK2 is a complex regulatory network that includes genes for replication and conjugal transfer, as well as for several potentially host-lethal proteins encoded by the kilA, kilB, and kilC loci. While kilB is known to be involved in conjugal transfer, the functions of kilA and kilC are unknown. The coregulation of kilA and kilC with replication and transfer genes indicates a possible role in the maintenance or broad host range of RK2. In this work, we found that a fourth kil locus, designated kilE, is located in the kb 2.4 to 4.5 region of RK2 and is regulated as part of the kil-kor regulon. The cloned kilE locus cannot be maintained in Escherichia coli host cells, unless korA or korC is also present in trans to control its expression. The nucleotide sequence of the kilE region revealed two potential multicistronic operons. The kleA operon consists of two genes, kleA and kleB, predicted to encode polypeptide products with molecular masses of 8.7 and 7.6 kDa, respectively. The kleC operon contains four genes, kleC, kleD, kleE, and kleF, with predicted products of 9.2, 8.0, 12.2, and 11.3 kDa, respectively. To identify the polypeptide products, each gene was cloned downstream of the phage T7 phi 10 promoter and expressed in vivo in the presence of T7 RNA polymerase. A polypeptide product of the expected size was observed for all six kle genes. In addition, kleF expressed a second polypeptide of 6 kDa that most likely results from the use of a predicted internal translational start site. The kleA and kleC genes are each preceded by sequences resembling strong sigma 70 promoters. Primer extension analysis revealed that the putative kleA and kleC promoters are functional in E. coli and that transcription is initiated at the expected nucleotides. The abundance of transcripts initiated in vivo from both the kleA and kleC promoters was reduced in cells containing korA or korC. When korA and korC were present together, they appeared to act synergistically in reducing the level of transcripts from both promoters. The kleA and kleC promoter regions are highly homologous and contain two palindromic sequences (A and C) that are the predicted targets for KorA and KorC proteins. DNA binding studies showed that protein extracts from korA-containing E. coli cells specifically retarded the electrophoretic mobility of DNA fragments containing palindrome A. Extracts from korC-containing cells altered the mobility of DNA fragments containing palindrome C. These results show that KorA and KorC both act as repressors of the kleAand kleC promoters. In the absence of korA and korC, expression of the cloned kleA operon was lethal to E.coli cells, whereas the cloned kleC operon gave rise to slowly growing, unhealthy colonies. Both phenotypes depended on at least one structural gene in each operon, suggesting that the operons encode genes whose products interact with critical host functions required for normal growth and viability. Thus, the kilA, kilC, and kilE loci of RK2 constitute a cluster of at least 10 genes that are coregulated with the plasmid replication initiator and the conjugal transfer system. Their potential toxicity to the host cell indicates that RK2 is able to establish a variety of intimate plasmid-host interactions that may be important to its survival in nature.

Amino Acid Sequence↗
PubMed1993

Four members of the maize beta-tubulin gene family are expressed in the male gametophyte.

Four different beta-tubulin cDNA clones have been identified in maize pollen cDNA libraries. Three of the four cDNA clones represent new maize beta-tubulin genes that have been designated tub3, tub4 and tub5. It is shown that the beta-tubulin gene family in maize is more complex than originally anticipated and more complex than those in vertebrates. In the maize beta-tubulin gene family the tub3, tub4 and tub5 genes are shown to represent small beta-tubulin gene subfamilies. Differences in the abundance of the tub3, tub4 and tub5 transcripts are seen among vegetative and reproductive tissues. The tub3 and tub4 transcripts are most abundant in pollen. In spikelet development, abundance of the tub3 transcript increases markedly from the 0.7 cm to the 1.0 cm spikelets whilst the levels of tub4 transcript closely resemble those of total beta-tubulin transcript in the organs examined. The tub3 and tub4 genes appear to have diverged recently in the evolution of the maize beta-tubulin gene family. The tub5 gene is expressed in pollen but its transcript is most abundant in maize vegetative tissues.

Amino Acid Sequence↗
PubMed2003

The CesA gene family of barley. Quantitative analysis of transcripts reveals two groups of co-expressed genes.

Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis.

Base Sequence↗
PubMed1997

A deletion mutation at the ep locus causes low seed coat peroxidase activity in soybean.

The Ep locus severely affects the amount of peroxidase enzyme in soybean seed coats. Plants containing the dominant Ep allele accumulate large amounts of peroxidase in the hourglass cells of the sub-epidermis. Homozygous recessive epep genotypes do not accumulate peroxidase in the hourglass cells and are much reduced in total seed coat peroxidase activity. To isolate the gene encoding the seed coat peroxidase and to determine whether it corresponds to the Ep locus, a cDNA library was constructed from developing seed coats and an abundant 1.3 kb peroxidase transcript was cloned. The corresponding structural gene was also isolated from a genomic library. Sequence analysis shows that the seed coat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa. Processing of a putative 26 amino acid signal sequence results in a mature protein of 326 residues with a calculated mass of 35 kDa and a pl of 4.4. Using probes derived from the cDNA, genomic DNA blot hybridization and polymerase chain reaction analysis detected polymorphisms that distinguished EpEp and epep genotypes. Co-segregation of the polymorphisms in an F2 population from a cross of EpEp and epep plants shows that the Ep locus encodes the seed coat peroxidase protein. Comparison of Ep and ep alleles indicates that the recessive gene lacks 87 bp of sequence encompassing the translation start codon. Analysis by RNA blot hybridization shows that epep plants have drastically reduced amounts of peroxidase transcript compared with EpEp plants. The peroxidase mRNA is abundant in seed coat tissues of EpEp plants during the late stages of seed maturation, and could also be detected in root tissues, but not in the flower, embryo, pod or leaf. The results indicate that the lack of peroxidase accumulation in seed coats of homozygous recessive epep plants is due to a mutation of the structural gene that reduces transcript abundance.

Amino Acid Sequence↗
PubMed2003

cDNA cloning, mRNA expression, and mutational analysis of the squalene synthase gene of Lotus japonicus.

A full-length cDNA for squalene synthase was isolated from Lotus japonicus, a model leguminous plant. The transcript was abundant in roots, symbiotic root nodules, and shoots, in that order. In situ hybridization revealed that the mRNA level is high in expanding root cells but low in dividing root tip ones. The transcript is also abundant in vascular bundles and the basal portions of mature nodules. L. japonicus squalene synthase has an unusual Asp residue near the active site, where mammalian enzymes have Gln, and replacement of the Gln by Glu has been reported to cause severe inactivation. Site-directed mutagenesis of the L. japonicus enzyme and assaying in vitro showed that this Asp residue can be substituted by not only Gln but also Glu, suggesting that the local structure of plant squalene synthases is different from that of mammalian enzymes.

Aspartic Acid↗