Northern Analysis Of Total RNA Research Articles

An Analysis of the Complete Genome Sequence and Subgenomic RNAs Reveals Unique Features of the Ampelovirus, Grapevine leafroll-associated virus 1

Despite being the first closterovirus documented in grapevines (Vitis sp.), the molecular biology of Grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus, family Closteroviridae) is still in its infancy. In this study, the complete genome sequence of two GLRaV-1 isolates was determined to be 18,731 (isolate WA-CH) and 18,946 (isolate WA-PN) nucleotides (nt). The genome of WA-CH and WA-PN isolates encodes nine putative open reading frames (ORFs) and the arrangement of these ORFs in both isolates was similar to that of Australian and Canadian isolates. In addition to two divergent copies of the coat protein (CP), the genome of GLRaV-1 isolates contain CP-homologous domain in four genes, making the virus unique among Closteroviridae members. The 5' and 3' nontranslated regions (NTRs) of WA-CH and WA-PN isolates showed differences in size and sequence composition, with 5' NTR having variable number of ∼65-nt-long repeats. Using the 5' NTR sequences, a reverse transcription-polymerase chain reaction and restriction fragment length polymorphism method was developed to distinguish GLRaV-1 variants in vineyards. Northern analysis of total RNA from GLRaV-1-infected grapevine samples revealed three subgenomic RNAs (sgRNAs), corresponding tentatively to CP, p21, and p24 ORFs, present at higher levels, with p24 sgRNA observed at relatively higher abundance than the other two sgRNAs. The 5' terminus of sgRNAs corresponding to CP, CPd1, CPd2, p21, and p24 were mapped to the virus genome and the leader sequence for these five sgRNAs determined to be 68, 27, 15, 49, and 18 nt, respectively. Taken together, this study provided a foundation for further elucidation of the comparative molecular biology of closteroviruses infecting grapevines.

Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme That Catalyzes Post-translational Protein Tyrosine O-Sulfation, Causes Male Infertility

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2(+/-) mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2(-/-) mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1(-/-) males that have normal fertility, Tpst2(-/-) males are infertile. Tpst2(-/-) sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2(-/-) sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility.

Cloning and Expression of a Xylitol-4-Dehydrogenase Gene from Pantoea ananatis

The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to L-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5' region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD+ as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the D-form of xylulose. Xylitol was converted to L-xylulose with a high yield (>80%) by the resting recombinant cells, and the L-xylulose was secreted into the medium. No evidence of D-xylulose being synthesized by the recombinant cells was found.

Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs.

The genome of hepatitis delta virus (HDV) is a small single-stranded circular RNA that is replicated via RNA-directed RNA synthesis. This makes use of a host RNA polymerase, probably pol II, that normally transcribes DNA templates. In vivo, the host polymerase can initiate replication from transfected linear RNAs using intramolecular template-switching. The present studies report that the polymerase could also achieve intermolecular switching leading to "reconstitution" of full-length HDV RNAs following transfection with two linear RNAs that were less than full length and yet lacking different regions of the genome. These two RNAs were synthesized in vitro, gel purified, pre-annealed, and then transfected into delta293, a cell line conditionally expressing the small delta antigen that is essential for HDV replication. Northern analyses of total RNA harvested from transfected cells detected the accumulation of full-length HDV genomic and antigenomic RNAs. Such reconstitution of full-length replicating HDV RNA was also achieved using nine other pairs of antigenomic RNAs and three pairs of genomic RNAs. Annealing of the RNAs prior to transfection was required for detectable HDV reconstitution. A second cell line, Huh7, also supported reconstitution when a pair of RNAs was cotransfected together with mRNA for the small delta protein. Taken together, these results support a model that observed genome reconstitution is a special form of recombination involving intermolecular template switches and they provide insights into the mechanism of RNA-directed RNA transcription catalyzed by a host RNA polymerase.

Altered gene expression in mouse livers after dichloroacetic acid exposure.

Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated that DCA exhibits hepatocarcinogenic effects in rodents when administered in drinking water. This chemical does not appear to be highly mutagenic, and the mechanism(s) involved in DCA induction of cancer are not clear. The present work was aimed at identifying changes in gene expression which may indicate critical alterations/pathways involved in this chemical's carcinogenic activities. We used cDNA microarray methods for analyses of gene expression in livers of mice treated with the tumorigenic dose of 2 g/l DCA in drinking water for 4 weeks. Total RNA samples obtained from livers of the control and DCA-treated mice were evaluated for gene expression patterns with Clontech Atlas Mouse 1.2 cDNA and Atlas mouse stress/toxicology arrays, and the data analyzed with AtlasImage 2.01 and one-way ANOVA in JMP4 software. From replicate experiments, we identified 24 genes with altered expression, of which 15 were confirmed by Northern blot analysis. Of the 15 genes, 14 revealed expression suppressed two- to five-fold; they included the following: MHR 23A, cytochrome P450 (CYP) 2C29, CYP 3A11, serum paraoxonase/arylesterase 1 (PON 1), liver carboxylesterase, alpha-1 antitrypsin, ER p72, glutathione S-transferase (GST) Pi 1, angiogenin, vitronectin precursor, cathepsin D (CTSD), plasminogen precursor (contains angiostatin), prothrombin precursor and integrin alpha 3 precursor (ITGA 3). An additional gene, CYP 2A4/5, had a two-fold elevation in expression. Further, in ancillary Northern analyses of total RNA isolated from DCA-induced hepatocellular carcinomas (from earlier reported studies of mice treated with 3.5 g/l DCA for 93 weeks), many of the same genes (11 of 15) noted above showed a similar alteration in expression. In summary, we have identified specific genes involved in the functional categories of cell growth, tissue remodeling, apoptosis, cancer progression and xenobiotic metabolism that have altered levels of expression following exposures to DCA. These findings serve to highlight new pathways in which to further probe DCA effects that may be critical to its tumorigenic activity.

Comparison of the variable loop regions of myosin heavy chain genes from Antarctic and temperate isopods

The evolutionary adaptations of functional genes to life at low temperatures are not well characterised in marine and fresh water invertebrates. Temperature has been shown to affect the functional characteristics of fish muscles, with changes in the velocity of shortening and ATPase activity being associated with myosin heavy chain (MyHC) isoform composition and the structure of the surface loop regions. Two PCR products spanning loops 1 and 2 of a MyHC gene from an Antarctic isopod ( Glyptonotus antarcticus) were sequenced and compared with those of a temperate isopod ( Idotea resecata), slow and fast fibres from lobster ( Homarus gammarus) and a cold water amphipod ( Eulimnogammarus verrucosus), revealing specific differences between the species, possibly related to fibre type and habitat temperature. The loop 2 region from G. antarcticus myosin was cloned and used for Northern analysis of total RNA from the other species. The cloned myosin cDNA hybridised specifically to a 6.6-kb transcript, in G. antarcticus muscle. In contrast, cDNA probes for lobster slow myosin and actin hybridised to muscle RNA from all species, demonstrating that a distinct MyHC isoform is expressed in the Antarctic isopod, as opposed to the temperate species. The inter- and intra-specific sequence differences in loop 2 region suggest that this may be a site for muscle adaptation to enable function at the low temperatures found in the Southern Ocean.

Rat costochondral chondrocytes produce 17β-estradiol and regulate its production by 1α,25(OH)2D3

Prior studies have shown that 17β-estradiol (17β-E2) regulates growth plate chondrocyte maturation and differentiation. This study examines the hypothesis that 17β-E2 is a local regulator of rat costochondral growth plate chondrocytes by determining whether these cells express aromatase mRNA and enzyme activity, produce 17β-E2, and regulate 17β-E2 production by vitamin D3 metabolites in a gender-specific and cell-maturation-dependent manner. Aromatase gene expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and northern analysis of total RNA from male and female chondrocytes. Aromatase specific activity was measured in cell layer lysates of confluent male and female rat costochondral resting zone (RC) and growth zone (GC) cartilage cells that had been treated for 24 h with 1α, 25(OH)2D3, 24R,25(OH)2D3, or transforming growth factor (TGF)-β1. 17β-E2 released into the culture media of treated cells was measured by radioimmunoassay (RIA). Female RC cells expressed the highest levels of aromatase mRNA compared with male RC cells and both male and female GC cells. Aromatase activity was present in male and female cells and was 1.6 times greater in female RC cells than female GC cells; male RC and GC cells displayed comparable levels. All cultures produced 17β-E2, with a 2.5-fold greater production by female RC cells than female GC cells or either cell type from male rats. Treatment of cultures with 1α,25(OH)2D3 caused a dose-dependent increase in 17β-E2 production by female RC (1.5-fold greater than control cells) and female GC (threefold greater than control cells) cells. In contrast, 1α,25(OH)2D3 had no effect on male GC cells and increased production in male RC cells by only 10% at the highest concentration of 1α,25(OH)2D3 used. Neither 24R, 25(OH)2D3 nor TGF-β1 had an effect on 17β -E2 production. These results support our hypothesis and indicate that 17β-E2 is most likely a local regulator of rat costochondral growth plate chondrocytes.

Expression of syndecan-1 during development, growth and cartilage degeneration in a transgenic mouse model for osteoarthritis

Mice heterozygous for the Del1 transgene locus with short deletion mutation in typeII collagen gene develop degenerative changes in the knee joints from the age of 3 months which progresses to an end-stage OA by the age of 12–15 months. This study focuses on expression and distribution in syndecan-1 during development of osteoarthritic cartilage degeneration. Human samples from cartilage of osteoarthritic patients was studied for comparison. Northern analysis of total RNA extracted from knee joints of transgenic Del1 mice and their nontransgenic controls was used to monitor changes in syndecan-1 levels during development, growth, aging and cartilage degeneration. Immunohistochemistry was used to study the distribution of syndecan-1 in mouse and human samples. Syndecan-1 was present in the knee joints during development, growth and aging both in the control and Del1 mice, the mRNA levels being highest in the aged and late osteoarthritic samples. The most intensive immunostaining of syndecan-1 was seen in synovial tissue and adjacent of the defected areas of cartilage and menisci. In addition, some individual cells or cell clusters in the superficial zone of articular cartilage contained syndecan-1. In human osteoarthritic cartilage, dedifferentiated syndecan-1 positive cells were seen in corresponding locations to those in Del1 mice. We demonstrated syndecan-1 for the first time in aging and degenerating murine articular cartilage and synovial tissue. Syndecan-1 is involved in phenotypic modulation of the articular chondrocytes and during osteophyte formation. In this Del1 mouse model, proliferation plays a role forming characteristic chondrocyte clusters near the surface, while apoptosis occurs primarily in the calcified cartilage. These results suggest that syndecan-1 has a role in the functional activity of the chondrocytes during the disease process. Control of syndecan expression in articular cartilage could be an attractive target for therapeutic interventions in the future.

Organisation and expression of the plastid genome of the dinoflagellate Amphidinium operculatum.

We show using PCR that psbC, atpA and petB genes are present in the plastid DNA minicircles from the dinoflagellate Amphidinium operculatum, extending the set of plastid genes identified from this organism. Unusually, the petBand atpA genes are located on the same minicircle. PCR using primers based on the "core" region found on all coding minicircles revealed the existence of a number of DNA minicircles with no apparent coding function. Northern analysis of total RNA from A. operculatum showed that the petB and atpA genes are represented on separate transcripts, despite being encoded in close proximity on the same minicircle. The possibility of transcript editing was investigated by RT-PCR, but psaA, psbA, psbB and atpB transcripts showed no evidence of editing, indicating that GUA can be used as an initiation codon in A. operculatum.

Cuticular Pro-phenoloxidase of the Silkworm, Bombyx mori: PURIFICATION AND DEMONSTRATION OF ITS TRANSPORT FROM HEMOLYMPH

Pro-phenoloxidase (proPO) in insects is implicated in the defense against microbes and wounding. The presence of proPO in the cuticle was suggested more than 30 years ago, but it has not been purified. The extract of cuticles of the silkworm, Bombyx mori, was shown to contain two proPO isoforms (F-type and S-type proPOs, which have slightly different mobilities in polyacrylamide gel electrophoresis under nondenaturing conditions). The two isoforms were purified to homogeneity. From hemolymph of the same insect, two types of proPO with the same electrophoretic mobilities as those of cuticular isoforms were separated and were shown to be different at five amino acid residues in one of their subunits. The isoforms in the hemolymph and cuticle were activated by a specific activating enzyme. The resulting active phenoloxidases exhibited almost the same substrate specificities and specific activities toward o-diphenols. The substrate specificities and the susceptibilities to inhibitors, including carbon monoxide, indicated that the purified proPO isoforms were not zymogens of laccase-type phenoloxidase. The proPO in hemolymph was shown to be transported to the cuticle. This demonstration was corroborated by the failure to detect proPO transcripts by Northern analysis of total RNA from epidermal cells. In reversed-phase column chromatography, cuticular and hemolymph proPOs gave distinct elution profiles, indicating that some yet to be identified modification occurs in hemolymph proPO and results in the formation of cuticular proPO. There was little transportation of cuticular proPO to the cuticle when it was injected into the hemocoel. The nature of the modification is described in the accompanying paper (Asano, T., and Ashida, M. (2001) J. Biol. Chem. 276, 11113-11125).

Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/Endoplasmic Reticulum Ca(2+)-ATPase.

The discontinuous pattern of muscle growth during the moulting cycle of a freshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). We describe the cloning, sequencing and characterization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abdominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). This complete sequence contains a 145 base pair (bp) noncoding region at the 5' end, a 3006 bp open reading frame coding for 1002 amino acid residues with a molecular mass of 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme contains all the conserved domains found in 'P'-type ATPases, and the hydropathy profile suggests a transmembrane organization typical of other SERCAs. It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% identity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitch muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle isoform SERCA2 and 67% identity with SERCA3. Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conserved and that most differences were in the NH(2) terminus, the central loop region and the COOH terminus. Northern analysis of total RNA from crayfish tissues probed with the 460 bp fragment initially isolated showed four bands (7.6, 7.0, 5.8 and 4.5 kilobases) displaying tissue-specific expression. SERCA was most abundant in muscle (axial abdominal, cardiac and stomach), where it is involved in Ca(2+) resequestration during relaxation, and in eggs, where it may be implicated in early embryogenesis. The level of SERCA mRNA expression in axial abdominal muscle varied during the moulting cycle as determined by slot-blot analysis. SERCA expression was greatest during intermoult and decreased to approximately 50% of this level during pre- and postmoult. Patterns of gene expression for SERCA and other sarcomeric proteins during the crustacean moulting cycle may be regulated by ecdysteroids and/or mechanical stimulation.

A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse.

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription-PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.

Breast tumour cell-induced down-regulation of type I collagen mRNA in fibroblasts.

This study investigated the modulation of type I collagen gene expression in normal fibroblasts by breast tumour cells. Northern analysis of total RNA extracted from stages I, II and III breast tumour tissue revealed that collagen mRNA levels were elevated in stage I tumours compared to the adjacent normal breast tissues, whereas they were decreased in stages II and III breast tumours. This aberrant collagen gene expression was confirmed by non-radioactive RNA:RNA in situ hybridization analysis of 30 breast carcinomas which localized the production of type I collagen mRNA to the stromal fibroblasts within the vicinity of the tumour cells. In order to determine whether the tumour cells were directly responsible for this altered collagen production by the adjacent fibroblasts, breast tumour cell lines were co-cultured with normal fibroblasts for in vitro assessment of collagen and steady-state collagen RNA levels. Co-culture of tumour cells and normal fibroblasts in the same dish resulted in down-regulation of collagen mRNA and protein. Treatment of the fibroblasts with tumour-cell conditioned medium also resulted in decreased collagen protein levels but the mRNA levels, however, remained unaltered. These results suggested that the tumour cells either secrete a labile ‘factor’, or express a cell surface protein requiring direct contact with the fibroblasts, resulting in down-regulation of collagen gene expression. Modulation of the ECM is a common characteristic of invading tumour cells and usually involves increased production of collagenases by the tumour cells or stromal fibroblasts. This study showed that tumour cells were also able to modulate collagen mRNA production by stromal fibroblasts, which may facilitate tumour cell invasion and metastasis. © 1999 Cancer Research Campaign

Partial synchronization of cell division in cultured maize (Zea mays L.) cells: differential cyclin, cdc2, histone, and retinoblastoma transcript accumulation during the cell cycle

Optimization of culture and treatment conditions for reproducible synchronization of the cell cycle in maize cells with hydroxyurea is described. Flow cytometric measurements of relative DNA contents revealed that, following the hydroxyurea block, around 55% of the cells were in the G1 phase (2C), 30% in the S phase (2C 4C) and 15% in the G2 phase (4C) of the cell cycle. The highest frequencies of cells in the S phase could be observed 2 h after removal of hydroxyurea (35-40%). The cells reached increased G2 phase frequencies (60-70%) between 8-14 h, while the maximum number of mitotic cells (12-14%) were found between 14-17 h. Northern analysis of total RNA from the synchronized cells indicated an increased level of transcripts from two histone (H3Zm, H4Zm) genes during the S-phase. The changes in the mRNA levels of the maize cyclin variants CycB1;zm;1, CycB1;zm;2 and CycA1;zm;1 support the classification of these cyclins as mitotic cyclins with transcript accumulation during the G 2 /M cell cycle phases. Using the maize retinoblastoma (ZmRb-1) cDNA as hybridization probe, two transcripts were detected with different hybridization intensity: the smaller, more abundant transcript was recognized during the whole cell cycle with an increase after the release of the cells from the hydroxyurea block, while the larger mRNA could only be detected for 8h after removal of hydroxyurea. The ZmRb-1 gene might respond to the increase of the frequency of cells in the S phase or to the re-addition of conditioned medium to the cells after hydroxyurea removal.

MRNA Expression of Renal Urea Transporters in Normal and Brattleboro Rats: Effect of Dietary Protein Intake

Differences in dietary protein level induce differences in fractional excretion of urea, in arginine vasopressin (AVP) plasma level, and in urine concentrating activity (in which intervene the renal urea transporters (UT)). The abundance of mRNA for UT-A1 (of the inner medullary collecting duct, IMCD) UT-A2 (of the descending thin limb) and UT-B1 (of descending vasa recta) was determined by Northern analysis of total RNA extracted from medullary subregions of Sprague-Dawley rats fed for 1 week, a low, normal, or high protein diet. The implication of AVP was then examined by studying AVP-deprived (Brattleboro) rats. Our results show that none of these transporters is affected by the level of protein intake, except UT-A1 that is reduced in terminal IMCD by low protein diet in the absence of AVP (Brattleboro rats). These data suggest that (1) the previously reported effect of kidney medulla hypertonicity on UT-A2 and UT-B1 mRNA expression is somehow obliterated by protein intake deficiency or excess, and (2) AVP influences the mRNA abundance of the UT-A1 of the terminal IMCD during protein deficiency.

Expression of proopiocortin and proopiomelanotropin during the life cycle of the sea lamprey (Petromyzon marinus)

The sea lamprey shows an important divergence in the proopiomelanocorticotropin (POMC) prohormone in that adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) are encoded on distinct genes, proopiocortin (POC) and proopiomelanotropin (POM), respectively. POC encodes nasohypophysial factor (NHF), ACTH, and β-endorphin while POM encodes α- and β-MSH-like peptides (called MSH-B and MSH-A, respectively), and a second form of β-endorphin. The present study examined the variation in expression of these two genes during the life cycle of sea lampreys. Individual Northern blot analysis and pooled data from several Northern blots showed significant differential expression of POC and POM during the lamprey life cycle. While POC expression remained low over larval and metamorphic stages, POM expression rose substantially in stage 4 and stage 6 of metamorphosis. Both POC and POM expression levels increased in immediately post-metamorphic animals. The highest levels of POM expression were observed in larger juvenile adults and pre-spawners, whereas the highest level of POC expression was observed at the pre-spawning period. Statistical analysis showed POM expression to be much higher than POC expression in immediately post-metamorphic animals prior to feeding. Northern analysis of total RNA isolated from various tissues in the pre-spawning lampreys, including brain, showed expression of POC and POM only in the pituitary. These observations indicate that the expression of POC and POM, which encode different sets of hormones, may be related to developmental and maturation events during the life cycle of the sea lamprey. J. Exp. Zool. 283:95–101, 1999. © 1999 Wiley-Liss, Inc.

Temporal and spatial patterns of accumulation of the transcript of Myo-inositol-1-phosphate synthase and phytin-containing particles during seed development in rice.

Myo-inositol-1-phosphate (I[1]P) synthase (EC 5.5.1.4) catalyzes the reaction from glucose 6-phosphate to I(1)P, the first step of myo-inositol biosynthesis. Among the metabolites of I(1)P is inositol hexakisphosphate, which forms a mixed salt called phytin or phytate, a storage form of phosphate and cations in seeds. We have isolated a rice (Oryza sativa L.) cDNA clone, pRINO1, that is highly homologous to the I(1)P synthase from yeast and plants. Northern analysis of total RNA showed that the transcript accumulated to high levels in embryos but was undetectable in shoots, roots, and flowers. In situ hybridization of developing seeds showed that the transcript first appeared in the apical region of globular-stage embryos 2 d after anthesis (DAA). Strong signals were detected in the scutellum and aleurone layer after 4 DAA. The level of the transcript in these cells increased until 7 DAA, after which time it gradually decreased. Phytin-containing particles called globoids appeared 4 DAA in the scutellum and aleurone layer, coinciding with the localization of the RINO1 transcript. The temporal and spatial patterns of accumulation of the RINO1 transcript and globoids suggest that I(1)P synthase directs phytin biosynthesis in rice seeds.

A high-density differential screening strategy for identification of fovea genes with altered expression in the retina during aging

A human fovea cDNA library was arrayed at high-density and reacted with age-specific (4 yr & 80 yr) retinal probes to identify genes with altered expression during aging. Using this approach, we screened 55,368 genes by Southern analysis and determined that ∼0.7% are differentially expressed in young and old tissues. Northern analysis of total RNA from retina of humans aged 2–94 yr show that expression of one of the clones identified (clone dd112) decreases with aging.

Cloning, genomic organization and chromosomal assignment of the mouse p190-B gene

The p190 family of GTPases consists of at least two different isoforms both containing an N-terminal GTPase and a C-terminal Rho GAP domain. Here we have isolated and characterized genomic and cDNA clones spanning the entire coding region of the mouse p190-B gene. Genomic data were obtained by sequencing plasmid subclones of two overlapping mouse genomic phage clones. Interestingly, a single 3.9 kb exon was found to contain approx. 80% of the coding region of the mouse p190-B protein (amino acid residues 1–1238) including the 5′-untranslated region, the N-terminal GTPase domain and a middle domain of unknown function. Missing from this exon, however, was the C-terminal Rho GAP domain, which was cloned from mouse brain mRNA using reverse transcriptase polymerase chain reaction. Comparison of the mouse with the human p190-B proteins revealed that approx. 97% of the amino acid residues were identical. Northern analysis of total RNA from a variety of mouse tissues detected ubiquitous expression of two p190-B transcripts of 4.0 and 6.8 kb in size. Analysis of two multilocus genetic crosses localized the mouse gene, Gfi2, to a position on chromosome 12, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 14. The high level of sequence homology between the human and the mouse suggests that there is a strong selective pressure to maintain the p190-B protein structure.

Thrombospondin-1 expression in human myometrium before and during pregnancy, before and during labor, and in human myometrial cells in culture.

The activation of latent transforming growth factor beta (L-TGFbeta) is essential for the action of TGFbeta, which, in turn, is involved in the regulation of expression of some progesterone-responsive genes. One mechanism by which TGFbeta is activated involves thrombospondin (TSP), a protein that binds extracellular proteins. Immunoreactive TSP (irTSP) protein and TSP-1 mRNA in myometrial tissues of ovulatory and pregnant women were localized by immunohistochemistry and in situ hybridization. IrTSP and TSP-1 mRNA were randomly distributed in myometrial smooth muscle cells of some, but not all, tissues of pregnant women at term before labor; but in some areas of most of these tissues, irTSP was intense and commonly localized extracellularly. Intense irTSP and TSP-1 mRNA in myocytes were more common in myometrium during labor. In myometrium from ovulatory women (n = 26), irTSP was localized primarily in vascular smooth muscle cells and was detected occasionally in scattered myocytes. Little TSP-1 mRNA was demonstrable by in situ hybridization in vessels or myocytes of myometrial tissue from ovulatory women (n = 7). By Northern analysis of total RNA, TSP-1 mRNA was detected in myometrial tissue of pregnant women and in human myometrial smooth muscle cells in culture. The levels of TSP-1 mRNA in myometrial tissues of pregnant women during labor (n = 18) were greater than those in myometrium at > 37 wk gestation before labor began (n = 25, p < 0.001). The ratios of TSP-1 to glyceraldehyde 3-phosphate dehydrogenase mRNAs in 3 myometrial tissues during oxytocin-induced labor were not statistically different from those in myometrium during spontaneous labor but were greater than those in myometrium before labor (p < 0.05). The level of TSP-1 mRNA in confluent human myometrial cells in culture was relatively high, was increased by treatment with fetal bovine serum, and was decreased by treatment with platelet-derived growth factor or activators of adenylyl cyclase or protein kinase C. Myometrial cells in culture constitute a useful model for studying the regulation of TSP-1 gene expression in human myometrium.