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At least 163 records · Page 9Linked to original sources
PubMed1999

Analysis of cDNA expressed sequence tags from Entamoeba histolytica: identification of two highly abundant polyadenylated transcripts with no overt open reading frames.

Upon analysis of 304 expressed sequence tags derived from the protozoan parasite Entamoeba histolytica, a number of novel protein encoding amoeba sequences were isolated. In addition, two unrelated, abundantly expressed transcripts were identified, and designated, ehapt1 and ehapt2. Although these transcripts do not contain any overt open reading frame, both are polyadenylated and together represent about 19% of total polyA+-RNA(11.6% for ehapt1 and 7.5% for ehapt2), thus being the most highly expressed polyA-containing transcripts so far identified in E. histolytica trophozoites. Northern blot and primer extension analyses revealed single-sized transcripts of 0.5 and 0.6 kb for ehapt1 and ehapt2, respectively, and Southern blot analysis suggests that both are encoded by multiple genes, which are distributed throughout the amoeba genome. Comparison between various ehapt1- and ehapt2-derived cDNAs indicated that both transcripts are highly polymorphic. Whereas nucleotide substitutions in ehapt2 are distributed throughout the sequence, variations in ehapt1 are mainly restricted to two regions, one of which comprises a deletion of variable length within an 8 nt tandem repeat unit. At present there is no convincing explanation for the possible role of ehapt1 and ehapt2 in E. histolytica, and analogous sequences have not been described so far for any other organism. Most likely they might represent regulatory RNAs or transcribed transposable elements.

Animals↗
PubMed2006

Molecular and biochemical analysis of periplastidial starch metabolism in the cryptophyte Guillardia theta.

Starch in synchronously grown Guillardia theta cells accumulates throughout the light phase, followed by a linear degradation during the night. In contrast to the case for other unicellular algae such as Chlamydomonas reinhardtii, no starch turnover occurred in this organism under continuous light. The gene encoding granule-bound starch synthase (GBSS1), the enzyme responsible for amylose synthesis, displays a diurnal expression cycle. The pattern consisted of a maximal transcript abundance around the middle of the light phase and a very low level during the night. This diurnal regulation of GBSS1 transcript abundance was demonstrated to be independent of the circadian clock but tightly light regulated. A similar yet opposite type of regulation pattern was found for two alpha-amylase isoforms and for one of the two plastidic triose phosphate transporter genes investigated. In these cases, however, the transcript abundance peaked in the night phase. The second plastidic triose phosphate transporter gene had the GBSS1 mRNA abundance pattern. Quantification of the GBSS1 activity revealed that not only gene expression but also total enzyme activity exhibited a maximum in the middle of the light phase. To gain a first insight into the transport processes involved in starch biosynthesis in cryptophytes, we demonstrated the presence of both plastidic triose phosphate transporter and plastidic ATP/ADP transporter activities in proteoliposomes harboring either total membranes or plastid envelope membranes from G. theta. These molecular and biochemical data are discussed with respect to the environmental conditions experienced by G. theta and with respect to the unique subcellular location of starch in cryptophytes.

Adenosine Triphosphate↗
PubMed1985

Apocytochrome b and other mitochondrial DNA sequences are differentially expressed during the life cycle of Trypanosoma brucei.

Cytochromes and Krebs cycle enzymes are not detected in bloodstream forms of Trypanosoma brucei but are present in procyclic forms. We have analyzed transcription of mitochondrial sequences which contain the apocytochrome b gene and several other open reading frames (ORFs). Multiple transcripts map to individual DNA sequences located on both DNA strands. Larger low abundance transcripts map to multiple ORFs and may be precursor RNAs. Small abundant transcripts map to G + C rich sequences that do not have obvious protein coding functions. The larger of two presumptive apocytochrome b transcripts is strikingly more abundant in procyclic than bloodstream forms and other mitochondrial transcripts are also differentially abundant between these two forms. In addition, many mitochondrial transcripts appear to be differentially polyadenylated between bloodstream and procyclic forms. We suggest that the mechanisms which regulate the production of the mitochondrial respiratory system in T. brucei involve differential expression of mitochondrial genes.

Animals↗
PubMed1996

Effect of CO2 Concentration on Carbonic Anhydrase and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Expression in Pea.

The effect of external CO2 concentration on the expression of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was examined in pea (Pisum sativum cv Little Marvel) leaves. Enzyme activities and their transcript levels were reduced in plants grown at 1000 [mu]L/L CO2 compared with plants grown in ambient air. Growth at 160 [mu]L/L CO2 also appeared to reduce steady-state transcript levels for rbcS, the gene encoding the small subunit of Rubisco, and for ca, the gene encoding CA; however, rbcS transcripts were reduced to a greater extent at this concentration. Rubisco activity was slightly lower in plants grown at 160 [mu]L/L CO2, and CA activity was significantly higher than that observed in air-grown plants. Transfer of plants from 1000 [mu]L/L to air levels of CO2 resulted in a rapid increase in both ca and rbcS transcript abundance in fully expanded leaves, followed by an increase in enzyme activity. Plants transferred from air to high-CO2 concentrations appeared to modulate transcript abundance and enzyme activity less quickly. Foliar carbohydrate levels were also examined in plants grown continuously at high and ambient CO2, and following changes in growth conditions that rapidly altered ca and rbcS transcript abundance and enzyme activities.

Journal Article↗
PubMed2002

Regulation of carbonic anhydrase gene expression in cotyledons of cotton (Gossypium hirsutum L.) seedlings during post-germinative growth.

Recently, plastidial carbonic anhydrase (CA) cDNA clones encoding functional carbonic anhydrase enzymes were isolated from a 48 h dark-grown cotton seedling (cotyledons) cDNA library (Hoang et al., Plant Cell Physiol. 40: 1999). Here we examined the levels of relative transcript abundance and enzyme activities in cotyledons at different developmental stages and under different environmental conditions (i.e. altering CO2 and light conditions), during post-germinative seedling growth. Relative CA transcript levels and total CA enzyme activity in cotyledons of cotton seedlings increased from 18 h to 72 h of post-germinative growth in the dark, although somewhat later than the glyoxylate cycle enzyme, MS. When 24 h old seedlings were exposed to light for an additional 24 h, CA activity in greening cotyledons increased about 2-fold (compared with controls kept in the dark), whereas relative CA transcript levels were essentially the same. Removal of seed coats from cotyledons of 24 h old seedlings dramatically increased relative CA transcript abundance (measured 24 h later) in the dark, but did not influence CA enzyme activity. Manipulation of external CO2 environments (zero, ambient, or high) modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activity. On the other hand, regardless of the external CO2 conditions, cotyledons of seedlings exposed to light exhibited increased CA activity, concomitant with increased Rubisco activity and increased chlorophyll content. Collectively, our data suggest that steady-state levels of CA and rbcS transcripts are increased in response to environmental CO2 conditions, while CA (and Rubisco) enzyme activities are likely modulated at the post-transcriptional level following exposure of seedlings to light, and in parallel with development of functional chloroplasts.

Blotting, Northern↗
PubMed2004

Dietary NaCl regulates renal aminopeptidase N: relevance to hypertension in the Dahl rat.

Aminopeptidase N (APN) is an abundant metallohydrolase in the brush border of kidney proximal tubule cells that degrades angiotensin III (Ang III) to angiotensin IV (Ang IV) and, along with dipeptidylaminopeptidase, degrades Ang IV. We examined the impact of a high-salt diet on renal APN activity and transcript abundance in the Sprague-Dawley and Dahl salt-sensitive (SS/Jr) rat strains. APN transcript abundance and protein abundance were approximately 2-fold greater (P<0.05; n=6) in the kidneys of Sprague-Dawley and Lewis rats ingesting 8% versus 0.3% salt diets, suggesting that increased aminopeptidase activity may contribute to decreased renal sodium uptake during adaptation to a high-salt diet. In contrast, renal APN transcript abundance and activity were the same in Dahl SS/Jr rats ingesting 8.0% versus 0.3% salt diets. The APN gene was mapped, using a radiation-hybrid panel, to known quantitative loci on chromosome 1 for blood pressure in the Dahl SS/Jr rat. The results suggest that the APN gene is a good candidate for salt-sensitivity in the Dahl SS/Jr rat.

Administration, Oral↗
PubMed1988

Directionality and further mapping of varicella zoster virus transcripts.

Our laboratory previously identified and preliminarily mapped 58 viral RNA transcripts in varicella zoster virus (VZV) infected cells (Ostrove et al., 1985). This study was initiated to more precisely map these transcripts, to identify additional transcripts, and to determine transcript directionality. To accomplish this, 32 overlapping BamHI, EcoRI, and SmaI fragments representing 99.7% of the genome were cloned into pGEM-2, a plasmid which contains a multiple cloning site flanked by SP6 and T7 RNA polymerase promoters. Each of these clones was used to produce 32P-labeled double-stranded DNA probes to detect transcripts homologous to either strand of the VZV insert, and single-stranded [32P]RNA probes in order to detect RNAs of either polarity. These probes were hybridized to Northern blots of VZV-infected cell RNA. In all, 77 RNAs were detected with both DNA and RNA probes. The direction of transcription and localization of 57 of the 58 previously identified RNAs and of 20 newly recognised abundant transcripts were determined. Thirty-three additional low-abundance transcripts were detected only by the relatively more sensitive RNA probes. A map indicating the directionality and approximate locations of the abundant VZV transcripts was constructed.

Chromosome Mapping↗
PubMed2002

Transcript profiling of human platelets using microarray and serial analysis of gene expression.

Human platelets are anucleate blood cells that retain cytoplasmic mRNA and maintain functionally intact protein translational capabilities. We have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of highly purified human blood platelets. Microarray analysis using the Affymetrix HG-U95Av2 approximately 12 600-probe set maximally identified the expression of 2147 (range, 13%-17%) platelet-expressed transcripts, with approximately 22% collectively involved in metabolism and receptor/signaling, and an overrepresentation of genes with unassigned function (32%). In contrast, a modified SAGE protocol using the Type IIS restriction enzyme MmeI (generating 21-base pair [bp] or 22-bp tags) demonstrated that 89% of tags represented mitochondrial (mt) transcripts (enriched in 16S and 12S ribosomal RNAs), presumably related to persistent mt-transcription in the absence of nuclear-derived transcripts. The frequency of non-mt SAGE tags paralleled average difference values (relative expression) for the most "abundant" transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet profiling. Quantitative reverse transcription-polymerase chain reaction (PCR) confirmed the highest frequency of mt-derived transcripts, along with the mRNAs for neurogranin (NGN, a protein kinase C substrate) and the complement lysis inhibitor clusterin among the top 5 most abundant transcripts. For confirmatory characterization, immunoblots and flow cytometric analyses were performed, establishing abundant cell-surface expression of clusterin and intracellular expression of NGN. These observations demonstrate a strong correlation between high transcript abundance and protein expression, and they establish the validity of transcript analysis as a tool for identifying novel platelet proteins that may regulate normal and pathologic platelet (and/or megakaryocyte) functions.

Base Sequence↗
PubMed2002

The abundant polyadenylated transcript 2 DNA sequence of the pathogenic protozoan parasite Entamoeba histolytica represents a nonautonomous non-long-terminal-repeat retrotransposon-like element which is absent in the closely related nonpathogenic species Entamoeba dispar.

While comparing gene expression in the pathogenic organism Entamoeba histolytica and the closely related but nonpathogenic species Entamoeba dispar, we discovered that the E. histolytica abundant polyadenylated transcript 2 (ehapt2) and corresponding genomic copies are absent in E. dispar. Although polyadenylated, ehapt2 does not contain any overt open reading frame. Southern blot and sequence analyses revealed that about 500 copies of ehapt2 genomic elements were present in each cell and that the copies were distributed throughout the ameba genome. The various ehapt2 elements are regularly located in the vicinity of protein-encoding genes, downstream of pyrimidine-rich sequence stretches (40 to 125 bp; CT content, 79.2 to 85.5%), and are flanked by duplicated target sites of variable length. Target site duplications were obviously generated during integration of ehapt2 into the E. histolytica genome as one copy of the flanking repeat and the complete ehapt2 element are specifically absent in orthologous E. dispar genomic sequences. ehapt2 shares 3' sequences with EhRLE, a recently identified non-long-terminal-repeat (non-LTR) retrotransposon-like element of E. histolytica, which contains a conceptual open reading frame for reverse transcriptase. Thus, ehapt2 has all of the properties of nonautonomous non-LTR retrotransposons. A comparison of various E. histolytica isolates suggested that transposition of ehapt2 takes place at a very low frequency as the genomic localization of ehapt2 elements was found to be well conserved. A mobile element such as ehapt2 could be a suitable mechanism to explain the infrequent and late transition of E. histolytica from a harmless gut commensal to an invasive pathogen.

Animals↗
PubMed1999

Thyroid hormone stimulates the renal Na/H exchanger NHE3 by transcriptional activation.

Thyroid hormone stimulates renal proximal tubule NaCl and NaHCO3 absorption in part by activating the apical membrane Na/H exchanger NHE3. We used a renal epithelial cell line, the opossum kidney (OK) cell, to define the mechanism by which 3,5,3'-triiodothyronine (T3) increases NHE3 activity. T3 stimulated NHE3 activity, an effect that was blocked by inhibition of cellular transcription or translation. The increase in activity was associated with increases in steady-state cell surface and total cellular NHE3 protein and NHE3 transcript abundance. T3 stimulated transcription of the NHE3 gene and had no effect on NHE3 transcript stability. The transcriptional activity of the 5'-flanking region of the rat NHE3 gene was stimulated by T3 when expressed in OK cells. When heterologously expressed rat NHE3 transcript levels were clamped constant with a constitutive promoter in OK cells, T3 has no effect on rat NHE3 protein abundance, suggesting the absence of regulation of NHE3 protein stability or translation. These studies demonstrate that T3 stimulates NHE3 activity by activating NHE3 gene transcription and increasing NHE3 transcript and protein abundance.

Animals↗
PubMed1996

Structure and characterization of a putative drought-inducible H1 histone gene.

A drought- and abscisic acid (ABA)-inducible gene, His1, was isolated from Lycopersicon pennellii, a drought-resistant relative of cultivated tomato, and the gene structure was defined experimentally. The nucleotide sequence of His1 predicts a protein of 202 amino acid residues, with a significant sequence homology to plant H1 histones. Consensus sequences for both H1 histone-specific promoter elements as well as an ABA-responsive element were identified in the 5'-flanking region of His1. Transcripts of this gene accumulate in leaf tissue in response to drought in three tomato species including cultivated tomato (L. esculentum), L. pennellii, and L. chilense, as well as in tobacco. Transcripts for His1 are constitutively expressed in roots; transcript abundances in tomato root tips were equivalent to transcript abundances in more mature regions of the seedling root. The accumulation in leaves of transcripts for His1 preceded visible symptoms of drought stress in the plants. Transcript accumulation was detected in both drought-sensitive and drought-resistant species at similar leaf water potentials, psi W -1.3 to -1.4 MPa.

Abscisic Acid↗
PubMed2000

The abundant larval transcript-1 and -2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis.

Lymphatic filariasis is a major tropical disease caused by the mosquito-borne nematodes Brugia and Wuchereria. About 120 million people are infected and at risk of lymphatic pathology such as acute lymphangitis and elephantiasis. Vaccines against filariasis must generate immunity to the infective mosquito-derived third-stage larva (L3) without accentuating immunopathogenic responses to lymphatic-dwelling adult parasites. We have identified two highly expressed genes, designated abundant larval transcript-1 and -2 (alt-1 and alt-2), from each of which mRNAs account for >1% of L3 cDNAs. ALT-1 and ALT-2 share 79% amino acid identity across 125 residues, including a putative signal sequence and a prominent acidic tract. Expression of alt-1 and alt-2 is initiated midway through development in the mosquito, peaking in the infective larva and declining sharply following entry into the host. Humans exposed to Brugia malayi show a high frequency of immunoglobulin G1 (IgG1) and IgG3 antibodies to ALT-1 and -2, distinguishing them from adult-stage antigens, which are targeted by the IgG4 isotype. Immunization of susceptible rodents (jirds) with ALT-1 elicited a 76% reduction in parasite survival, the highest reported for a single antigen from any filarial parasite. ALT-1 and the closely related ALT-2 are therefore strong candidates for a future vaccine against human filariasis.

Amino Acid Sequence↗
PubMed2026

Norgestrel drives mitochondrial collapse and plasma membrane impairment in Pacific oyster (Crassostrea gigas) sperm by triggering premature acrosome reaction.

The toxic mechanisms of norgestrel (NGT), an emerging marine pollutant, on the sperm from externally fertilized invertebrates remain elusive. This study employed an integrated physiological and multi-omics framework to elucidate how NGT (10 and 1000&#xa0;ng/L) disrupts acrosome reaction (AR) signaling machinery, thereby impairing the functional integrity of Pacific oyster (Crassostrea gigas, also known as Magallana gigas) sperm. Exposure to NGT triggered a significant, dose-dependent premature AR, characterized by elevated acrosin activity and a loss of acrosomal integrity. Multi-omics integration supports a model in which this premature exocytosis is linked to signaling disturbances, including disruption of calcium signaling and reduced transcript abundance of calmodulin (CaM) and the primary recognition protein zonadhesin (Zan). This signaling interference induced an premature AR, subsequently driving a cascade of bioenergetic and structural failures. At the mitochondrial level, NGT induced abnormal mitochondrial permeability transition pore (mPTP) opening and elevated the transcript levels of antioxidant defense genes (e.g., peroxiredoxin-5, PRDX5). These alterations indicate the occurrence of mitochondrial collapse. Concurrently, scanning electron microscopy verified localized plasma membrane wrinkling and pore formation in sperm. In addition, NGT exposure decreased the transcript abundance of cytoskeleton-related genes, including solute carrier family 26 member 6 (SLC26A6), actin (ACT), and tubulin polymerization promoting protein family member 3 (TPPP3). These molecular changes further disrupted membrane phospholipid homeostasis, as represented by altered glycerophospholipid metabolism. At the same time, cumulative cellular stress was associated with decreased transcript abundance of cytoprotective factors (e.g., baculoviral IAP repeat-containing proteins, birc2) and changes in apoptosis-related genes consistent with activation of a caspase-8-mediated apoptotic programme. In conclusion, NGT, as a representative synthetic progestin, exerts reproductive toxicity by interfering with signaling mediators to induce premature AR, which subsequently exhausts metabolic energy and triggers plasma membrane impairment. These findings provide a critical mechanistic basis for the aquatic ecological risk assessment of synthetic progestins.

Animals↗
PubMed2006

Staying alive in adversity: transcriptome dynamics in the stress-resistant dauer larva.

In response to food depletion and overcrowding, the soil nematode Caenorhabditis elegans can arrest development and form an alternate third larval stage called the dauer. Though nonfeeding, the dauer larva is long lived and stress resistant. Metabolic and transcription rates are lowered but the transcriptome of the dauer is complex. In this study, distribution analysis of transcript profiles generated by Serial Analysis of Gene Expression (SAGE) in dauer larvae and in mixed developmental stages is presented. An inverse relationship was observed between frequency and abundance/copy number of SAGE tag types (transcripts) in both profiles. In the dauer profile, a relatively greater proportion of highly abundant transcripts was counterbalanced by a smaller fraction of low to moderately abundant transcripts. Comparisons of abundant tag counts between the two profiles revealed relative enrichment in the dauer profile of transcripts with predicted or known involvement in ribosome biogenesis and protein synthesis, membrane transport, and immune responses. Translation-coupled mRNA decay is proposed as part of an immune-like stress response in the dauer larva. An influence of genomic region on transcript level may reflect the coordination of transcription and mRNA turnover.

Acclimatization↗
PubMed1999

Insulin-glucocorticoid interactions in the regulation of acetyl-CoA carboxylase-alpha transcript diversity in ovine adipose tissue.

Transcription of the acetyl-CoA carboxylase (ACC)-alpha gene is initiated from two promoters, promoter I (PI) and promoter II (PII) such that transcripts demonstrate heterogeneity in their 5' untranslated regions (UTR). Exons 1 and 2 (E1 and E2) are the primary exons in transcripts initiated from PI and PII respectively; E5 is the first coding exon present in all transcripts. In addition alternative exon splicing results in transcripts that either include or exclude a 47 nucleotide sequence corresponding to E4, such that E[1/4/5] and E[1/5] type transcripts result from PI activity, whereas transcripts containing E[2/4/5] or E[2/5] in the 5'UTR result from PII. In subcutaneous adipose tissue from non-pregnant non-lactating sheep approximately 60% of ACC-alpha transcripts are derived from PI, of which 85% are the E[1/5] type. Lactation resulted in an 88% reduction in total PI transcripts, of which the E[1/5] type was reduced 90% and the E[1/4/5] type 80%. By contrast lactation reduced the total levels of PII transcripts by only 50%. Culture of explants from the subcutaneous depot of lactating sheep with insulin plus dexamethasone for 72 h resulted in an 8-fold increase in both E[1/4/5] and E[1/5] types when compared with explants prior to culture. PII transcripts, by contrast, were increased 2-fold by culture in insulin plus dexamethasone and this was entirely attributed to an increase in the expression of the E[2/4/5] type. Dexamethasone acts to potentiate the action of insulin on PI and PII transcript abundance and this effect is greatest for PI transcripts. This study has demonstrated that repression of the ACC-alpha gene in adipose tissue during lactation is largely achieved through attenuation of PI transcript abundance and may be related, in part, to a change in the sensitivity of the apparatus that regulates PI transcript steady-state levels to insulin.

Acetyl-CoA Carboxylase↗
PubMed2005

Comparison of Helicobacter pylori virulence gene expression in vitro and in the Rhesus macaque.

We used a quantitative real-time reverse transcriptase PCR assay to measure the transcript abundance of 46 known and putative Helicobacter pylori virulence genes, including 24 genes on the Cag pathogenicity island. The expression profile of H. pylori cells grown in vitro was also compared to expression in vivo after experimental infection of rhesus macaques. Transcript abundance in vitro (mid-log phase) ranged from about 0.004 (feoB and hpaA) to 20 (ureAB, napA, and cag25) copies/cell. Expression of most genes was repressed during the transition from logarithmic- to stationary-phase growth, but several well-characterized H. pylori virulence genes (katA, napA, vacA, and cagA) were induced. Comparison of results in the rhesus macaque with similar data from humans showed a strong correlation (r = 0.89). The relative in vivo expression in the rhesus monkey was highly correlated with in vitro expression during mid-log (r = 0.89)- and stationary (r = 0.88)-phase growth. Transcript abundance was on average three- to fourfold reduced in vivo compared to in vitro during mid-log phase. However, when compared to stationary phase, increased expression in vivo was observed for 6 of 7 genes on a contiguous portion of the pathogenicity island, several of which are thought to encode the H. pylori type IV structural pilus and its accessory proteins. These results suggest the possibility that some genes encoding the H. pylori type IV structural pilus and accessory proteins may form an operon that is induced during growth in vivo.

Animals↗
PubMed2005

Alloplasmic effects on mitochondrial transcriptional activity and RNA turnover result in accumulated transcripts of Arabidopsis orfs in cytoplasmic male-sterile Brassica napus.

Mitochondrial transcription was investigated in a cytoplasmic male-sterile (CMS) Brassica napus line with rearranged mitochondrial (mt) DNA mostly inherited from Arabidopsis thaliana. The transcript patterns were compared with the corresponding male-fertile progenitors, B. napus and A. thaliana, and a fertility-restored line. Transcriptional activities, gene stoichiometry and transcript steady-state levels were analysed for all protein and rRNA coding genes and for several orfs present in the A. thaliana mitochondrial genome. The transcriptional activities were highly variable when comparing the parental species, while the CMS and restored lines displayed similar activities. For several ribosomal protein genes transcriptional activity was reduced while it was increased for orf139 in comparison with the parental species. The differences in transcriptional activity observed could be related to differences in relative promoter strength, as gene stoichiometry between lines was very limited. Transcript steady-state levels were more homogenous than the transcriptional activities demonstrating RNA turnover as a compensating mechanism. In the CMS line higher transcript abundance and novel transcript patterns in comparison with the parental lines were found for several genes. Of those, the transcripts for orf139, orf240a and orf294 were less abundant in the fertility-restored line. These putative CMS-associated transcripts were mapped by cRT-PCR. In conclusion we show that (mt) DNA from A. thaliana was non-correctly transcribed and processed/degraded in the B. napus nuclear background. Furthermore, the introgressed nuclear A. thaliana DNA in the fertility-restored line contributes to a more rapid degradation of transcripts accumulated from A. thaliana derived orfs in the CMS line.

Arabidopsis↗
PubMed1994

Microdissected double-minute DNA detects variable patterns of chromosomal localizations and multiple abundantly expressed transcripts in normal and leukemic cells.

Double-minute (dm) chromosomes are cytogenetically resolvable DNA amplification-mediating acentric extrachromosomal structures that are commonly seen in primary tumors, tumor cell lines, and drug-resistant cells grown in vitro. Selective isolation of dm DNAs with standard molecular biological techniques is difficult, and thus, detailed studies to elucidate their structure, site of chromosomal origin, and chromosomal reintegration patterns have been limited. In those instances in which a gene has been localized on dms, characterization of the remainder of the DNA, which far exceeds the size of the gene identified, has remained inconclusive, dms seen in the acute myeloid leukemia cell line HL-60 have been shown to harbor the c-myc protooncogene. In this paper, we report the successful isolation of the dm-specific DNAs from these cells by the microdissection/polymerase chain reaction technique and demonstrate that the dm DNAs derived from a single discrete normal chromosome segment 8q24.1-q24.2 reintegrate at various specific locations in the leukemic cells. The microdissected dm DNA detects multiple abundantly expressed transcripts distinct from c-myc mRNA on Northern blots. By devising a "transcript selection" strategy, we cloned the partial genomic sequence of a gene from the microdissected DNA that encodes two of these RNAs. This strategy will be generally applicable for rapid cloning of unknown amplified genes harbored on dms. With DNA from 20 microdissected dms, we constructed a genomic library of about 20,000 recombinant microclones with an average insert size of about 450 bp.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗