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Cell-Specific Expression of Mitochondrial Transcripts in Maize Seedlings.

Although mitochondria are thought to assume crucial and possibly novel physiological functions during male gametogenesis, it is not known to what extent mitochondrial function is necessary for other aspects of plant development or to what degree the expression of plant mitochondrial genes is subject to cell-specific regulation, particularly during vegetative growth. We have used in situ hybridization to show that extensive differences exist in the levels of mitochondrial RNAs (mtRNAs) among different tissues and among different individual cell types within the same organ of maize seedlings. The expression of all examined mtRNAs is enhanced in vascular bundles, particularly in procambium- and xylem-forming cells. Mitochondrial transcript levels correlated highly with cell division activity. For example, in roots, the transcripts are abundant in the dividing cells of the meristem but drop to very low levels in the nondividing cells of the root cap and the meristem quiescent center. By comparison, levels of functional mitochondria, as assessed by rhodamine-123 fluorescence, did not vary greatly among the same group of cells. In shoots, in situ hybridization and blot hybridization revealed differences in the patterns of localization among different mtRNAs. The results indicate that during vegetative growth, mitochondrial gene expression at the transcript level is subject to an unexpected degree of cell-specific regulation and that different controls may operate on different trancripts.

Journal Article↗

Trefoil factor family (TFF) expression in the mouse brain and pituitary: changes in the developing cerebellum.

Trefoil factor family (TFF) peptides, besides their prominent expression in mucous epithelia, are also synthesized in the central nervous system. Previously TFF1 expression was observed in mouse brain astrocytes, while oxytocinergic neurons of the hypothalamo-pituitary axis are recognized sites of TFF3 synthesis. Here, the expression of TFF1, TFF2, and TFF3 was systematically studied using reverse transcription-polymerase chain reaction (RT-PCR) analysis of dissected adult mouse brain regions including the pituitary. Additionally, the developmental profile of TFF expression in murine cerebral cortex and cerebellum was monitored. Overall, the expression patterns of the three TFF genes differed. The TFF1 and TFF2 profiles shared some similarities, whereas the TFF3 expression pattern was completely different. TFF1 was nearly uniformly, but weakly expressed in all brain regions tested. The TFF1 and TFF2 expression patterns differed characteristically in the pituitary where abundant TFF2 transcription was detected in the anterior and not the posterior lobe and the expression level in males was higher than in females. In contrast, TFF3 expression was limited to the hippocampus, the temporal cortex, and the cerebellum, the latter being surprisingly the major site of expression. Here, TFF3 mRNA appeared to be restricted mainly to neurons and not glial cells. Cerebellar TFF3 expression is clearly developmentally regulated (maximum at P15), indicating a role for TFF3 during postnatal cerebellar development.

Animals↗

mRNA transcripts initiating within the human immunoglobulin mu heavy chain enhancer region contain a non-translatable exon and are extremely heterogeneous at the 5' end.

Transcription events are thought to precede gene rearrangement in the immunoglobulin (Ig) loci and may be the mechanism by which the various gene regions are made accessible for recombination. If this is the case, identification and characterization of transcripts from the Ig loci should permit a better understanding of the gene rearrangement process. We have isolated a 2.3 kb cDNA clone from the human pre-B cell line Nalm-1 that contains enhancer-specific sequences from the Ig heavy (H) chain gene locus. The 2.3 kb transcript initiated within the enhancer region and showed extreme 5' heterogeneity, with more than 50 initiation sites mapping near the Ig-specific octamer ATTTGCGT. Sequencing of the cDNA clone demonstrated that 644 nucleotides from the Ig enhancer region were incorporated as a leader exon spliced to the mu constant (Cmu) region. This leader exon contained many translation termination codons and may function to inhibit the translation of sterile Cmu polypeptides. Using an enhancer-derived probe, we detected two low-abundancy mRNA transcripts with sizes of 2.3 and 12 kb. Northern blot analysis suggested that the 12 kb transcript was the unspliced precursor mRNA of a VDJ rearrangement. The potential role of these enhancer-containing transcripts in the opening of the IgH chain gene for rearrangement and for class switching is discussed.

Amino Acid Sequence↗

Aberrant transcripts of the FHIT gene are expressed in normal and leukaemic haemopoietic cells.

Deletions and apparent transcriptional abnormalities of the FHIT gene at 3p14.2 have recently been reported in a wide variety of solid tumours. To determine whether lesions of this gene also occur in leukaemia, we have analysed a total of 97 patients (chronic myeloid leukaemia, CML, in chronic phase or blast crisis, n = 71; de novo acute leukaemia, n = 26) and 16 normal individuals. Intact FHIT transcripts from all cases were amplified using RT-PCR. In addition, smaller size bands that were less intense than the full-length products were amplified from several samples from patients with leukaemia and also from normal leucocytes. Sequencing of the small products revealed that they were derived from FHIT transcripts lacking whole exons. Using single-strand conformation polymorphism analysis, no mutations in the coding sequence were detected in any patient. Furthermore, loss of heterozygosity was not seen in any of 36 informative patients at D3S1300 or D3S1481, markers located within the FHIT locus. We conclude that the FHIT gene and other uncharacterized tumour-suppressor genes at 3p14.2 are unlikely to be involved in the pathogenesis of acute leukaemia or progression of CML from chronic phase to blast crisis. Moreover, low-abundance FHIT transcripts that lack whole exons are not specific to malignant cells and should not be taken as evidence of an abnormality in the absence of demonstrable genomic DNA lesions.

Acid Anhydride Hydrolases↗

Origin of the mRNA stoichiometry of the puf operon in Rhodobacter sphaeroides.

The LH-I structural genes are located 5' of the RC-L and -M structural genes on what has been designated as the puf operon of Rhodobacter sphaeroides. Analysis of puf operon expression in R. sphaeroides by Northern hybridization with probes specific for individual structural gene has identified two transcripts encoded by this operon. The large (2.6 kilobase pairs (kb] transcript contains sequences for all four polypeptides of the puf operon, whereas the small (0.5 kb) transcript, which is more abundant (10-15-fold) than the large transcript under photosynthetic growth, is homologous only to the two LH-I structural genes. Transcription of the puf operon during photosynthetic growth under saturating light conditions is increased approximately 3-fold relative to growth in the presence of oxygen while the relative ratio of these two transcripts is independent of the incident light intensity. Analysis of the turnover of the two transcripts (t1/2 of 9 and 20 min for the large and small transcripts, respectively) indicates that 5' processing is the initial step in the degradation of the large transcript and that the molar excess of the small transcript cannot be accounted for by differences in the rates of turnover of these two mRNA species. Analysis of the 5' ends of the 2.6- and 0.5-kb transcripts, their relative abundance, and stabilities indicates that these two transcripts have different 5'-ends corresponding to 75 and 104 base pairs upstream from the start of the LH-I beta structural gene, respectively. Northern hybridization analysis with specific synthetic deoxyoligonucleotide probes confirmed that the two transcripts differ by 29 bases at their 5'-ends, suggesting that differential transcript initiation may be involved in regulating the relative levels of these two mRNA species in vivo, although we cannot rule out complex mechanisms of post-transcriptional processing.

Bacterial Proteins↗

Role of the bga1-encoded extracellular {beta}-galactosidase of Hypocrea jecorina in cellulase induction by lactose.

Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major beta-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular beta-galactosidase during growth on lactose. Its transcript was abundant during growth on l-arabinose and l-arabinitol but was much less common when the organism was grown on lactose, d-galactose, galactitol, d-xylose, and xylitol. Deltabga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular beta-galactosidase activity that was not encoded by bga1 but no intracellular beta-galactosidase activity. In conclusion, cellulase production on lactose occurs when beta-galactosidase activity levels are low but decreases as the beta-galactosidase activities increase. The data indicate that bga1-encoded beta-galactosidase activity is a critical factor for cellulase production on lactose.

Cellulase↗

Molecular cloning and characterization of human cardiac homeobox gene CSX1.

Accumulating evidence has suggested that homeo-domain-containing proteins play critical roles in regulating the tissue-specific gene expression essential for tissue differentiation and in determining the temporal and spatial patterns of development. In order to elucidate the mechanisms of human heart development, we have isolated a human homologue of the murine cardiac homeobox gene Csx (also called Nkx-2.5) and denoted it as CSX1. The amino acid sequence of the CSX1 homeodomain is 100% and 67% identical to that of murine Csx/Nkx-2.5 and Drosophila tinman, respectively. CSX1 has at least three isoforms generated by an alternative splicing mechanism. One of these isoforms (CSX1a) encodes a protein of approximately 35 kD that possesses the homeodomain, whereas the other two (CSX1b and CSX1c) encode a truncated protein of approximately 12 kD that is identical to the CSX1a protein at the amino-terminal 112 amino acids but lacks the homeodomain. Northern blot analysis showed that CSX1 transcripts are abundantly expressed in both fetal and adult hearts, but no signal was detected in other human tissues examined. Amplification of each isoform by reverse transcriptase-polymerase chain reaction revealed that all of the three isoforms are expressed in fetal and adult hearts and that the homeobox-containing isoform CSX1a is most abundant. The homeodomain-containing protein encoded by CSX1a binds to Csx/Nkx-2.5 binding sequences and transactivates the sequence-containing luciferase reporter gene. Unexpectedly, the homeodomain-lacking protein encoded by CSX1b also transactivates the reporter gene, although CSX1b does not bind to the Csx/Nkx-2.5 binding sequences. The highly conserved homeodomain sequence in evolution and the restricted expression in the heart suggest that CSX1 plays an important role in the development and differentiation of the human heart and that there may be two different mechanisms in transcriptional regulation by the CSX1 protein, homeodomain-dependent and -independent mechanisms.

Base Sequence↗

The S3a ribosomal protein gene is identical to the Fte-1 (v-fos transformation effector) gene and the TNF-alpha-induced TU-11 gene, and its transcript level is altered in transformed and tumor cells.

Previous work on mouse x rat hybrid cells (BS series) led to the assignment of a transformation suppressor locus (Sail) to the rat 5q22-q33 region. This gene is not yet identified. From a non-transformed BS hybrid cell line, we isolated a partial cDNA insert (13T), which detects a transcript more abundant in transformed cells than in their non-transformed homologs. Sequence comparisons led us to conclude that 13T is identical to the coding sequences of the ribosomal protein S3a gene (Rps3a), of Fte-1 (v-fos transformation effector gene) and of TU-11, a mouse gene induced by TNF-alpha. Rps3a, Fte-1 and TU-11 are thus one and the same gene. Similarity was also found between this gene and non-mammalian sequences reported to be involved in cell cycling. Like the Rps3a transcript level, the c-Fos transcript level is higher in transformed cells. Rps3a and Fos could thus be effectors of the transformed phenotype.

Animals↗

Sequencing and analysis of common bean ESTs. Building a foundation for functional genomics.

Although common bean (Phaseolus vulgaris) is the most important grain legume in the developing world for human consumption, few genomic resources exist for this species. The objectives of this research were to develop expressed sequence tag (EST) resources for common bean and assess nodule gene expression through high-density macroarrays. We sequenced a total of 21,026 ESTs derived from 5 different cDNA libraries, including nitrogen-fixing root nodules, phosphorus-deficient roots, developing pods, and leaves of the Mesoamerican genotype, Negro Jamapa 81. The fifth source of ESTs was a leaf cDNA library derived from the Andean genotype, G19833. Of the total high-quality sequences, 5,703 ESTs were classified as singletons, while 10,078 were assembled into 2,226 contigs producing a nonredundant set of 7,969 different transcripts. Sequences were grouped according to 4 main categories, metabolism (34%), cell cycle and plant development (11%), interaction with the environment (19%), and unknown function (36%), and further subdivided into 15 subcategories. Comparisons to other legume EST projects suggest that an entirely different repertoire of genes is expressed in common bean nodules. Phaseolus-specific contigs, gene families, and single nucleotide polymorphisms were also identified from the EST collection. Functional aspects of individual bean organs were reflected by the 20 contigs from each library composed of the most redundant ESTs. The abundance of transcripts corresponding to selected contigs was evaluated by RNA blots to determine whether gene expression determined by laboratory methods correlated with in silico expression. Evaluation of root nodule gene expression by macroarrays and RNA blots showed that genes related to nitrogen and carbon metabolism are integrated for ureide production. Resources developed in this project provide genetic and genomic tools for an international consortium devoted to bean improvement.

Carbon↗

Inhibition by recombinant human interleukin-6 of the glucagon-dependent induction of phosphoenolpyruvate carboxykinase and of the insulin-dependent induction of glucokinase gene expression in cultured rat hepatocytes: regulation of gene transcription and messenger RNA degradation.

The influence of recombinant human interleukin-6, the major mediator of the inflammatory response in liver, on the glucagon- and insulin-dependent induction of the phosphoenolpyruvate carboxykinase and glucokinase gene, respectively, was monitored on the level of gene transcription, mRNA abundance and enzyme activity in cultured rat hepatocytes. As control markers of the interleukin-6-induced acute-phase response the mRNA levels of the acute phase proteins alpha 2-macroglobulin and beta-fibrinogen were determined. In cultured rat hepatocytes, recombinant human interleukin-6, added simultaneously with glucagon and insulin, lowered the maximal increase in glucagon-induced phosphoenolpyruvate carboxykinase mRNA levels after 2 hr and the maximal increase in glucokinase mRNA levels after 3 hr to about 30%, respectively. It inhibited the glucagon-induced increase in phosphoenolpyruvate carboxykinase gene transcription and phosphoenolpyruvate carboxykinase enzyme activity, as well as the insulin-induced increases in glucokinase gene transcription and glucokinase enzyme activity. Recombinant human interleukin-6 increased the mRNA levels of the acute-phase proteins alpha 2-macroglobulin and beta-fibrinogen gradually over 4 to 6 hr. Recombinant human interleukin-6, added 2 hr after glucagon or 3 hr after insulin at the maximum of the hormone-induced enzyme mRNA levels, almost doubled the decay rate of phosphoenolpyruvate carboxykinase mRNA and glucokinase mRNA. The results show that interleukin-6 induced the expression of inflammatory proteins and simultaneously inhibited the hormone-induced expression of enzymes of intermediary metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Species- and tissue-specific transcription of complex, highly repeated satellite-like Bsp elements in the fox genome.

We have studied the transcription of highly repeated satellite-like Bsp elements containing the potential promoter boxes for RNA polymerase III in the genomes of adult silver and arctic foxes. The Bsp repeat transcripts were abundant enough to be detected by Northern blot and semiquantitative dot blot hybridizations, and the majority were found in the nuclear RNA fraction from arctic fox kidneys. Weak hybridization signals were revealed with the cytoplasmic RNA preparation from silver fox kidneys and with the nuclear RNA fraction from arctic fox liver, and their intensity was intermediate with the total RNA from arctic fox brain. Taken together, the data suggest possible genomic interspersion of some Bsp repeats in these two representatives of Canidae. The observed species-and tissue-specificity of the transcription of Bsp repeats suggests that they may potentially accomplish regulatory functions in the fox genomes.

Animals↗

The MyoD binding site is dispensable for cardiac actin gene expression in the somites of later stage Xenopus embryos.

We have studied the transcription directed by the promoter of a Xenopus borealis cardiac actin gene fused to a globin reporter sequence after injection into X. laevis embryos. This promoter is known to be effective specifically in somites of neurula stage embryos, and work on a very similar X. laevis gene has indicated the importance of an upstream MyoD binding site (E box) in this process. We show that, although in the absence of the MyoD site there is a small amount of tissue-specific expression in neurulae, transcription is abundant in the tailbud embryo a few hours later. Thus, although the E box is initially essential, other sequences must adopt the same role soon afterwards.

Actins↗

Quantification of transcripts from the ICP4 and thymidine kinase genes in mouse ganglia latently infected with herpes simplex virus.

Herpes simplex virus establishes latency in nervous tissue in which it is maintained for the life of the mammalian host, with occasional reactivation leading to subsequent spread. Latency-associated transcripts are abundant during latency, but viral proteins and productive cycle RNAs have not been detected. Using sensitive, quantitative PCR assays, we have quantified certain viral RNAs specific to productive-cycle genes in mouse ganglia latently infected with herpes simplex virus type 1. Sense-strand RNA specific to the essential immediate-early gene, ICP4, was present in most ganglia in variable amounts relative to the amount of viral DNA, with one to seven molecules of RNA per viral genome in about 20% of ganglia. In contrast, the amount of latency-associated transcripts was much less variable, at an average of 4 x 10(4) molecules per viral genome. The amounts of ICP4-specific RNA were similar at 30 and 60 days postinfection, and at least some of these transcripts initiated within a region consistent with utilization of the ICP4 promoter. RNA specific to the thymidine kinase gene, whose transcription in productive infection is dependent on ICP4, was present in latently infected ganglia at a maximum level of 3.2 x 10(6) molecules per ganglion (500 molecules per viral genome). ICP4-specific and tk-specific RNAs measured from the same samples showed a positive correlation extending over 2 orders of magnitude. We conclude that ICP4-specific RNA is expressed in the absence of detectable reactivation and discuss possible implications of our findings for latent gene expression.

Animals↗

A desiccation-related Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA.

The resurrection plant Craterostigma plantagineum tolerates an extreme loss of cellular water. Therefore this plant is being studied as model system to analyse desiccation tolerance at the molecular level. Upon dehydration, new transcripts are abundantly expressed in different tissues of the plant. One such desiccation-related nuclear gene (dsp-22 for desiccation stress protein) encodes a mature 21 kDa protein which accumulates in the chloroplasts. Sequence analysis indicates that dsp-22 is closely related to early light inducible genes (Elip) of higher plants and to a carotene biosynthesis related gene (cbr) isolated from the green alga Dunaliella bardawil. In contrast to other desiccation-related genes, light is an essential positive factor regulating the expression of dsp-22: ABA-mediated gene activation leads to the accumulation of the transcript only in the presence of light. During the desiccation process, light acts at the transcriptional and post-transcriptional levels. The implications of these different controls and the possible role of the dsp-22 protein in the desiccation/rehydration process are discussed.

Abscisic Acid↗

Transcriptional control of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme in transgenic mice: role for nuclear receptors in cardiac and brown adipose expression.

Expression of the gene encoding medium-chain acyl coenzyme A dehydrogenase (MCAD), a nuclearly encoded mitochondrial fatty acid beta-oxidation enzyme, is regulated in parallel with fatty acid oxidation rates among tissues and during development. We have shown previously that the human MCAD gene promoter contains a pleiotropic element (nuclear receptor response element [NRRE-1]) that confers transcriptional activation or repression by members of the nuclear receptor superfamily. Mice transgenic for human MCAD gene promoter fragments fused to a chloramphenicol acetyltransferase gene reporter were produced and characterized to evaluate the role of NRRE-1 and other promoter elements in the transcriptional control of the MCAD gene in vivo. Expression of the full-length MCAD promoter-chloramphenicol acetyltransferase transgene (MCADCAT.371) paralleled the known tissue-specific differences in mitochondrial beta-oxidation rates and MCAD expression. MCADCAT.371 transcripts were abundant in heart tissue and brown adipose tissue, tissues with high-level MCAD expression. During perinatal cardiac developmental stages, expression of the MCADCAT.371 transgene paralleled mouse MCAD mRNA levels. In contrast, expression of a mutant MCADCAT transgene, which lacked NRRE-1 (MCADCATdeltaNRRE-1), was not enriched in heart or brown adipose tissue and did not exhibit appropriate postnatal induction in the developing heart. Transient-transfection studies with MCAD promoter-luciferase constructs containing normal or mutant NRRE-1 sequences demonstrated that the nuclear receptor binding sequences within NRRE-1 are necessary for high-level transcriptional activity in primary rat cardiocytes. Electrophoretic mobility shift assays demonstrated that NRRE-1 was bound by several cardiac and brown adipose nuclear proteins and that these interactions required the NRRE-1 receptor binding hexamer sequences. Antibody supershift studies identified the orphan nuclear receptor COUP-TF as one of the endogenous cardiac proteins which bound NRRE-1. These results dictate an important role for nuclear receptors in the transcriptional control of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme and identify a gene regulatory pathway involved in cardiac energy metabolism.

Acyl-CoA Dehydrogenase↗

Cytoplasmic localization of transcripts of a complex G+C-rich crab satellite DNA.

The primary sequence and higher order structures of a G+C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis have been described previously. The repeat unit of the satellite is approximately 2.1 kb. In exploring a possible function for this satellite, we asked whether it is transcribed. As a probe for transcripts, we used a segment of DNA amplified from a 368 bp EcoRI fragment from the very highly conserved 3' end of the satellite DNA. During polymerase chain reaction (PCR) amplification, the probe was simultaneously either radiolabeled or biotinylated. Tissue- and stage-specific transcripts were observed when blots of poly(A)+ mRNAs recovered from polysomes isolated from crab tissues [including midgut gland (hepatopancreas), limb bud, and claw muscle] were probed with the satellite DNA fragment. The presence of satellite transcripts in polysomal mRNAs is strong evidence that the transcripts had reached the cytoplasm. To corroborate the presence of transcripts in the cytoplasm, we investigated in situ hybridization of satellite probes with RNAs in tissue sections. Biotinylated satellite DNA probes were applied to sections of midgut gland, limb bud papilla, ovary, or testis of anecdysial crabs. Retention of RNAs in tissue sections was improved by UV-irradiation prior to hybridization. Transcripts were abundant in the cytoplasm of all tissues except testis. Sections of crab midgut gland treated with RNase A prior to hybridization and sections of mouse pancreatic tumor served as controls; neither showed any signals with the probe.

Animals↗

Mechanism of action of antisense RNA. Sometime inhibition of transcription, processing, transport, or translation.

Anyone considering the use of AS RNA, generated endogenously, to inhibit gene expression should plan to generate several independent transfectants with nonoverlapping sequences; strategies that maximize both the transcription rate and the stability of the AS RNA are obviously desirable. Reasons why different results are obtained in different systems or with different constructs likely include the specific nucleotide sequence under investigation, the location of the AS gene in the nucleus relative to the endogenous gene, and the rate-limiting step in the expression of the target gene. Splicing may not be necessary, but an efficient polyadenylation signal likely is. Employment of a ribozyme-mediated strategy, discussed by various investigators in this volume, may be beneficial. There is no reason at present to conclude that any gene, however abundant its transcript might be, is inherently recalcitrant to AS-mediated down-regulation of expression.

Biological Transport↗

Alpha1,3-fucosyltransferase IX (Fuc-TIX) is very highly conserved between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc-TIX.

The amino acid sequence of Fuc-TIX is very highly conserved between mouse and human. The number of non-synonymous nucleotide substitutions of the Fuc-TIX gene between human and mouse was strikingly low, and almost equivalent to that of the alpha-actin gene. This indicates that Fuc-TIX is under a strong selective pressure of preservation during evolution. The human Fuc-TIX (hFuc-TIX) showed a unique characteristics, i.e. hFuc-TIX was not activated by Mn2+ and Co2+, whereas hFuc-TIV and hFuc-TVI were activated by the cations. The hFuc-TIX transcripts were abundantly expressed in brain and stomach, and interestingly were detected in spleen and peripheral blood leukocytes.

Amino Acid Sequence↗