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Introduction: Turner syndrome (TS) is a chromosomal disorder that affects phenotypic females who have one intact X chromosome and complete or partial absence of the second sex chromosome in association with one or more clinical manifestations. However, the immunological profile of TS with different X chromosome origins is incompletely understood., Methods: In this study, transcriptomic expression profiles of 26 TS (45,X) samples and 10 normal karyotype (46,XX) samples derived from GSE46687 cohort were employed. Differentially expressed immune-related genes (DEIRGs) between monosomy X TS patients with different X chromosome origins and normal females were investigated respectively. Subsequently, functional annotation, protein-protein interaction (PPI) network analysis, immunocyte infiltration evaluation, tissue-specific gene expression and Weighted gene co expression network analysis (WGCNA) were performed to explore the immunological characteristic in TS with different X chromosome origins., Results: 34 and 52 DEIRGs were respectively identified in 45,Xm and 45,Xp patients compared with normal individuals. The identified DEIRGs in Xm group were significantly enriched in pathways associated with cancer. In Xp TS patients, the most enriched signals were immune response-related. A majority of genes involved in the above pathways were downregulated. PPI analysis identified 4 ( FLT3, IL3RA, CSF2RA, PIK3R3 ) and 6 ( PDGFRB , CSF2 , IL5 , PRL , CCL17 and IL2 )hub genes for Xm and Xp groups, respectively. CIBERSORT results showed that the proportion of Tregs in the Xm group and the naive B cells and resting NK cells in the Xp group significantly increased, respectively. Tissue-specific expression results indicated that BDCA4+_dentritic cells and CD19+ B cells were the prominent specific expressed tissues in Xp patients. Results of WGCNA support the above analysis., Conclusions: This study aims at studying the immunological characteristics of TS with different X chromosome origins. Pathways in cancer in Xm group and immune response in Xp group were suppressed. 4 and 6 hub IRGs were identified as biomarkers for Xm and Xp patients, respectively. B cells played important roles in Xp patients. Further studies are needed to draw more attention to the functional validation of these hub genes and the roles of B cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Qi, Wang, Yu, Kong, Shi and Wang.)

The micropylar region of endosperm (ME) is a physical barrier to radicle emergence in seeds of many different species, including tomato (Solanum lycopersicum) and Arabidopsis thaliana. ME is thought to be weakened through cell wall-modifying proteins, and this is supported by transcriptome data showing enrichment of cell wall-associated genes in ME. Gibberellin and ethylene have been suggested to be involved in induction of these genes in ME. However, mechanisms underlying this critical event for germination still remain elusive. In addition to hormonal regulation of ME weakening, recent data from high-throughput analyses suggested that it might be important for the radicle tip to ‘touch’ ME (or mechanosensing), in terms of ME-specific gene induction. This emerging hypothesis can be integrated with previous hypotheses about hormonal regulation of ME-specific gene expression in seeds. [ABSTRACT FROM PUBLISHER]

The physiological functions of a tissue in the body are carried out by its complement of expressed genes. Genes that execute a particular function should be more specifically expressed in tissues that perform the function. Given this premise, we mined public microarray expression data to build a database of genes ranked by their specificity of expression in multiple organs. The database permitted the accurate identification of genes and functions known to be specific to individual organs. Next, we used the database to predict transcriptional regulators of brown adipose tissue (BAT) and validated two candidate genes. Based upon hypotheses regarding pathways shared between combinations of BAT or white adipose tissue (WAT) and other organs, we identified genes that met threshold criteria for specific or counterspecific expression in each tissue. By contrasting WAT to the heart and BAT, the two most mitochondria-rich tissues in the body, we discovered a novel function for the transcription factor ESRRG in the induction of BAT genes in white adipocytes. Because the heart and other estrogen-related receptor gamma (ESRRG)-rich tissues do not express BAT markers, we hypothesized that an adipocyte coregulator acts with ESRRG. By comparing WAT and BAT to the heart, brain, kidney and skeletal muscle, we discovered that an isoform of the transcription factor sterol regulatory element binding transcription factor 1 (SREBF1) induces BAT markers in C2C12 myocytes in the presence of ESRRG. The results demonstrate a straightforward bioinformatic strategy to associate genes with functions. The database upon which the strategy is based is provided so that investigators can perform their own screens. [ABSTRACT FROM AUTHOR]

This article discusses embryonic stem (ES) cells. The use and knowledge of the ES cells nowadays are mentioned as coming from earlier works on cancer cells involving mice by, among others, Leroy Stevens at the Jackson Labs and Kleinsmith and Pierce. The early beginning of transgenic technology in the early 1970s is related. The article also addresses the partial replacement of ES cell technology by zinc finger nuclease technology and looks as well into the future of mutant mouse.

This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism. [ABSTRACT FROM PUBLISHER]

Reduced availability of water for agricultural use has been forecast for much of the planet as a result of global warming and greater urban demand for water in large metropolitan areas. Strategic improvement of water use efficiency (WUE) and drought tolerance in perennial crops, like fruit trees, could reduce water use without compromising yield or quality. We studied water use in apple trees using 'Royal Gala', a relatively water use-efficient cultivar, as a standard. To examine whether genes useful for improving WUE are represented in a wild relative genetically close to M. xdomestica, we surveyed Malus sieversii for traits associated with WUE and drought resistance using material collected from xeric sites in Kazakhstan. This collection has been maintained in Geneva, NY, and surveyed for various phenotypes and has been genetically characterized using simple sequence repeats (SSRs). These data suggest that most of the diversity in this population is contained within a subpopulation of 34 individuals. Analysis of this subpopulation for morphological traits traditionally associated with WUE or drought resistance, e.g., leaf size and stomata size and arrangement, indicated that these traits were not substantially different. These results imply that some of the genetic diversity may be associated with changes in the biochemistry, uptake, and/or transport of water, carbon, or oxygen that have allowed these trees to survive in water-limited environments. Furthermore, genes responding to drought treatment were isolated from 'Royal Gala' and categorized according to the biological processes with which they are associated. A large fraction of upregulated genes from roots were identified as stress-responsive, whereas genes from leaves were for the most part associated with photosynthesis. We plan to examine expression of these genes in the M. sieversii population during water deficit in future studies to compare their patterns of expression with 'Royal Gala'. [ABSTRACT FROM AUTHOR]

Abstract: To understand the tissue-specific expression of the rat placental lactogen-I variant (rPL-Iv) gene, we investigated the methylation pattern of the 5′-flanking region of this gene in various rat tissues. We report that the 5′-flanking region of the rPL-Iv gene was hypomethylated in placenta that expressed the gene and hypermethylated in those tissues that did not express the gene. Moreover, the intron region of the rPL-Iv gene was hypomethylated in the placenta, but hypermethylated in the liver, kidney and pituitary. Although there are 5 CpG sites and the density of CpG dinucleotide is lower within 2 kb of the rPL-Iv 5′-flanking region, the methylated promoter reporter gene produced strong repression in the transcriptional activity of the gene. In addition, the 5′-flanking and intron regions of the rPL-Iv gene were hypomethylated on day 12 of gestation, and the methylation pattern in the placenta remained unchanged from mid-pregnancy until term. The entire genomic region of the rPL-Iv gene might be hypermethylated in tissues other than the placenta, within which its methylated status repress expression of the placenta-specific rPL-Iv gene. Interestingly, the methylation status of the intron region of the rPL-Iv in proliferating Rcho-1 cells was changed to the unmethylated status on day 8 and 12 of differentiation of Rcho-1 cells. These results demonstrate that demethylation in the rPL-Iv upstream region was induced at an early stage of placental development, and once the 5′-flanking region of the rPL-Iv had been demethylated, its status on the rPL-Iv genomic region was continued during pregnancy. Taken together, these results suggest that DNA methylation is responsible for the silencing of tissue-specific genes in non-expressing cells, while defined combinations of trophoblast factors dictate the expression of unmethylated rPL-Iv gene in placenta trophoblast cells. [ABSTRACT FROM AUTHOR]

Human organic cation transporter 2 (OCT2/SLC22A2), which is specifically expressed in the kidney, plays critical roles in the renal secretion of cationic compounds. Tissue expression and membrane localization of OCT2 are closely related to the tissue distribution, pharmacological effects, and/or adverse effects of its substrate drugs. However, the molecular mechanisms underlying the kidney-specific expression of OCT2 have not been elucidated. In the present study, therefore, we examined the contribution of DNA methylation of the promoter region for the OCT2 gene to its tissue-specific expression using human tissue samples. In vivo methylation status of the proximal promoter region of OCT2 and that of OCT1, a liver-specific organic cation transporter, were investigated by bisulfite sequencing using human genomic DNA extracted from the kidney and liver. All CpG sites in the OCT2 proximal promoter were hypermethylated in the liver, while hypomethylated in the kidney. On the other hand, the promoter region of OCT1 was hypermethylated in both the kidney and liver. The level of methylation of the OCT2 promoter was especially low at the CpG site in the E-box, the binding site of the basal transcription factor upstream stimulating factor (USF) 1. In vitro methylation of the OCT2 proximal promoter dramatically reduced the transcriptional activity, and an electrophoretic mobility shift assay showed that methylation at the E-box inhibited the binding of USF1. These results indicate that kidney-specific expression of human OCT2 is regulated by methylation of the proximal promoter region, interfering with the transactivation by USF1. [ABSTRACT FROM AUTHOR]

Abstract: It has been postulated that noncoding RNAs (ncRNAs) are involved in the posttranscriptional control of gene expression, and may have contributed to the emergence of the complex attributes observed in mammalians. We show here that the complement of ncRNAs expressed from intronic regions of the human and mouse genomes comprises at least 78,147 and 39,660 transcriptional units, respectively. To identify conserved intronic sequences expressed in both humans and mice, we used custom-designed human cDNA microarrays to separately interrogate RNA from mouse and human liver, kidney, and prostate tissues. An overlapping tissue expression signature was detected for both species, comprising 198 transcripts; among these, 22 RNAs map to intronic regions with evidence of evolutionary conservation in humans and mice. Transcription of selected human–mouse intronic ncRNAs was confirmed using strand-specific RT-PCR. Altogether, these results support an evolutionarily conserved role of intronic ncRNAs in human and mouse, which are likely to be involved in the fine tuning of gene expression regulation in different mammalian tissues. [Copyright &y& Elsevier]

Opalin is a transmembrane protein detected specifically in mammalian oligodendrocytes. Opalin homologs are found only in mammals and not in the genome sequences of other animal classes. We first determined the nucleotide sequences of Opalin orthologs and their flanking regions derived from four prosimians, a group of primitive primates. A global comparison revealed that an evolutionarily conserved region exists in the first intron of Opalin. When the conserved domain was assayed for its enhancer activity in transgenic mice, oligodendrocyte-directed expression was observed. In an oligodendroglial cell line, Oli-neu, the conserved domain showed oligodendrocyte-directed expression. The conserved domain is composed of eight subdomains, some of which contain binding sites for Myt1 and cAMP-response element binding protein (CREB). Deletion analysis and cotransfection experiments revealed that the subdomains have critical roles in Opalin gene expression. Over-expression of Myt1, treatment of the cell with leukemia inhibitory factor (LIF), and cAMP analog (CREB activator) enhanced the expression of endogenous Opalin in Oli-neu cells and activated the oligodendrocyte enhancer. These results suggest that LIF, cAMP signaling cascades and Myt1 play significant roles in the differentiation of oligodendrocytes through their action on the Opalin oligodendrocyte enhancer. [ABSTRACT FROM AUTHOR]

Maf family transcription factors are regulators of tissue-specific gene expression and cell-differentiation in a wide variety of tissues and are also involved in human diseases and oncogenic transformation. To establish tissue-specific expression, Maf binds to Maf-recognition elements (MAREs) in the regulatory regions of target genes, and functionally interacts with other transcription factors. For example, L-Maf and c-Maf, which are specifically expressed in developing lens cells, act synergistically with Sox proteins to induce lens-specific crystalline genes. MafA, a β-cell-specific member of the Maf family, activates the insulin gene promoter synergistically with Pdx1 and Beta2 to establish β-cell specific expression. Furthermore, in β-cells, MafA activity is regulated at both the transcriptional and post-translational levels by glucose and oxidative stress. This review summarizes the functions and roles of Maf in various biological processes and recent progress in elucidating the mechanisms whereby Maf proteins regulate transcription. [ABSTRACT FROM PUBLISHER]

Abstract: Based on bioinformatics analysis, we previously hypothesized the existence of a bipartite TDPOZ protein family members of which carry the TRAF domain (TD) and POZ/BTB [Huang, C.-J., Chen, C.-Y., Chen, H.-H., Tsai, S.-F., Choo, K.-B., 2004. TDPOZ, a family of bipartite animal and plant proteins that contain the TRAF (TD) and POZ/BTB domains. Gene 324, 117–127.]. Conservation in animals and plants suggests important biological functions for the putative TDPOZ proteins. In this work, we report testis-specific expression of two new Tdpoz members, Rtdpoz-T1 and -T2, of the rat genome; the result clearly indicates that members of the hypothetical gene family are, indeed, expressed. T1 and T2 cDNA sequences were derived by rapid amplification of cDNA ends (RACE). The exons of the genes were determined by queries of the rat genome sequence draft and selectively confirmed in splicing assays. The results indicate that T1 and T2 share a common leader exon indicative of alternative splicing, and that the genes are uninterrupted by introns in their respective coding sequences. Database interrogations also reveal a combined 297 hits of Rtdpoz-like sequences on 7 chromosomes; however, the bulk of the hits (264) and 26 putative TDPOZ-encoding genes, including T1 and T2, are found in a ∼2.5 Mb cluster in the Rn2_2148 supercontig on chromosome 2. Our data signify retrotransposition in the generation and expansion of the Rtdpoz repertoire in the rat genome. We also anticipate spatio-temporal-specific expression of many more TDPOZ members in the rat or other animals and plants. [Copyright &y& Elsevier]

Here we report the selective expression of two POU transcription factor genes, PLA-1 and OCT-1, in human placenta and choriocarcinoma cell lines JAR, JEG-3 and BeWo. Pla-1 protein binds to a POU-consensus DNA sequence in the human placental lactogen-3 (PL-3) promoter and it is capable of trans-activating its transcription up to 18-fold. Other tissue-specific or ubiquitous POU transcription factors such as Pit-1/GHF-1 or Oct-1 showed none or low levels of trans-activation of the PL-3 promoter. In addition, we identified an unique and highly charged region in the N-terminal portion of Pla-1 protein required for full trans-activation of the PL-3 promoter. [Copyright &y& Elsevier]

The somatic cells of a multicellular organism contain an identical complement of genes that need to be expressed specifically and appropriately to allow the normal development and functions associated with an organism. In the eukaryotic cell nucleus, genes are packaged with nucleoprotein histones into chromatin. The human growth hormone (GH)/chorionic somatomammotropin (CS) gene family offers an excellent model to study the relationship between chromatin structure and transcription factor binding in terms of tissue-specific gene expression. The GH/CS gene family consists of five genes (GH-N, GH-V, CS-A, CS-B and CS-L), contained in a single locus on chromosome 17. Although they share approximately 94% sequence similarity, GH-N expression is restricted to pituitary somatotropes while the four placental GH/CS genes are expressed in the villus syncytiotrophoblast. Appropriate expression in vivo is dependent on remote sequences found 14–32 kb upstream of GH-N in the loci of adjacent genes, and these sequences are characterized by five (I–V) nuclease-hypersensitive sites (HS). Pituitary-specific factor Pit-1 binds at HS I/II and plays an essential role in chromatin remodeling and GH-N expression; however, the processes that lead to HS I/II accessibility are unknown. We discuss the possibility that Pit-1-driven remodeling at HS III may precede that at HS I/II in the pituitary. Also, in pituitary chromatin, all five GH/CS genes share similar nuclease sensitivity, suggesting that the conformation of the placental genes is not inhibitory to transcription. Given that the promoters of both GH-N and the placental GH/CS genes contain Pit-1-binding sites, possible mechanisms to restrict placenta GH/CS promoter activity in the pituitary are discussed, including active repression via P sequences located upstream of each of the placental GH/CS genes. Positively or negatively influencing those components known to be important for pituitary transcription may link epigenetic events to key transcription factors in the overall picture of tissue-specific control of gene expression. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Specific expression of the insulin gene in pancreatic islet β-cells requires multiple cis-regulatory elements in its promoter. Pdx1, MafA, and Beta2 have been identified as β-cell enriched transcription factors that bind to these elements. Pdx1 has been shown to bind to A1, A3, A5, and GG2, and Beta2 binds to E1 by forming a heterodimer with the ubiquitous factor E47. MafA was recently identified as a C1-element binding factor. However, interactions between these factors and the promoter have not been characterized in detail. In this report, we show that these transactivators synergistically stimulate insulin promoter activity. Among multiple binding sites for Pdx1, MafA, and Beta2, at least GG2, C1, and E1 elements located in the promoter region between −150 and −100 base pairs are necessary for the synergism. We also found that neither MafB nor c-Maf, close relatives of MafA, showed synergistic activation. These results suggest that co-expression and functional synergism of these β-cell enriched transactivators, MafA, Pdx1, and Beta2, are critical for establishing the β-cell-specific and efficient expression of the insulin gene. [Copyright &y& Elsevier]

Powdery mildew is an important disease of wheat caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. This pathogen invades exclusively epidermal cells after penetrating directly through the cell wall. Because powdery mildew colonizes exclusively epidermal cells, it is of importance not only to identify genes which are activated, but also to monitor tissue specificity of gene activation. Acquired resistance of wheat to powdery mildew can be induced by a previous inoculation with the non-host pathogen B. graminis f. sp. hordei, the causal agent of barley powdery mildew. The establishment of the resistant state is accompanied by the activation of genes. Here we report the tissue-specific cDNA-AFLP analysis and cloning of transcripts accumulating 6 and 24 h after the resistance-inducing inoculation with B. graminis f. sp. hordei. A total of 25 000 fragments estimated to represent about 17 000 transcripts were displayed. Out of these, 141 transcripts, were found to accumulate after Bgh inoculation using microarray hybridization analysis. Forty-four accumulated predominantly in the epidermis whereas 76 transcripts accumulated mostly in mesophyll tissue. [ABSTRACT FROM AUTHOR]

The mammalian olfactory mucosa (OM) is unique among extrahepatic tissues in having high levels, and tissue-selective forms, of cytochrome P450 (CYP) enzymes. These enzymes may have important toxicological implications, as well as biological functions, in this chemosensory organ. In addition to a tissue-selective, abundant expression of CYP1A2, CYP2A, and CYP2G1, some of the OM CYPs are also known to have an early developmental expression, a resistance to xenobiotic inducers, and a lack of responsiveness to circadian rhythm. Efforts to fully characterize the regulation of CYP expression in the OM, and to identify the underlying mechanisms, are important for our understanding of the physiological functions and toxicological significance of these biotransformation enzymes, and may also shed unique light on the general mechanisms of CYP regulation. The aim of this mini-review is to provide a summary of current knowledge of the various modes of regulation of CYPs expressed in the OM, an update on our mechanistic studies on tissue-selective CYP expression, and a review of the literature on xenobiotic inducibility of OM CYPs. Our goal is to stimulate further studies in this exciting research area, which is of considerable importance, in view of the constant exposure of the human nasal tissues to inhaled, as well as systemically derived, chemicals, the prevalence of olfactory system damage in individuals with neurodegenerative diseases, and the current uncertainty in risk assessments for potential olfactory toxicants. [Copyright &y& Elsevier]

To achieve the liver-directed expression in sufficient amounts of therapeutic genes for successful and safe gene therapy, natural liver-specific promoters can be used to direct the expression of therapeutic genes in the liver, whereas strong viral enhancers were used to obtain sufficient amounts of expressed therapeutic gene products. However, very often use of either the former or the latter does not guarantee both potent and liver-specific therapeutic gene expression. Here we conglomerate them and thus create a potent tissue-specific promoter by characterizing and using the liver-type pyruvate kinase proximal promoter (LPKPP) harboring its TATA box and a HNF-1α binding site. Alone it hardly activated its reporter gene expression in non-hepatocytes or hepatocytes. However, in the presence of the SV40 viral enhancer (SV40VE), which is active in multiple cell types, it was able to potently activate its reporter gene expression specifically in hepatocytes. The tissue-specific activation of the LPKPP by the viral enhancer was attributed to HNF-1α binding to the LPKPP. Taken together, these results support the idea that the constitutively active SV40VE could be used to activate the LPKPP in a tissue-specific manner in the presence of HNF-1α. To our knowledge, this is the first study to utilize HNF-1α and its binding site, in the context of the LPKPP, to generate a basal promoter that is transcriptionally activated potently in a tissue-specific manner by a viral enhancer that is active in multiple cell types. [Copyright &y& Elsevier]

Slitrk family proteins are characterized as integral membrane proteins that have two leucine-rich repeat (LRR) domains and a carboxy-terminal domain that is partially similar to trk neurotrophin receptor proteins. The LRR domains are similar to those of slit proteins. In a previous study, we showed that mouse Slitrk genes are expressed predominantly in neural tissue and have neurite-modulating activity in cultured neuronal cells. Their expression profiles as well as their functions vary among the family members. In this paper, we characterized the human SLITRK1, SLITRK2, SLITRK3, SLITRK4, SLITRK5, and SLITRK6 genes. The six genes are located in three clusters, on 3q, 13q, and Xq, respectively. Their expression was detected mainly in the brain, but the expression profile of each SLITRK was unique. SLITRK expression was also investigated in various types of brain tumor tissue. The results showed that all SLITRK genes are differentially expressed in brain tumors, including astrocytoma, oligodendroglioma, glioblastoma, medulloblastoma, and supratentorial primitive neuroectodermal tumor (PNET). Particularly interesting findings were that SLITRK3 expression was enhanced in tissue from several different types of tumors and SLITRK6 expression was highly selective. These results suggest that the human SLITRK genes are useful molecular indicators of brain tumor properties. [Copyright &y& Elsevier]

In an attempt to understand the role of gene-associated CpG islands in the expression of plant genes, I determined the position of CpG islands within their associated genes and the expression of the genes in rice tissues. I examined the expression patterns of 75 rice genes by Northern hybridization analysis using RNAs isolated from four rice tissues: leaf, root, callus, and panicle at flowering stage. From the results of this analysis, I classified most of the genes into one of two groups: expression in a single tissue and expression in two or more tissues. There was a marked correlation between the expression of a gene in two or more tissues and the presence of a CpG island in its 5′-end (class 1 CpG island). Among the genes expressed in a single tissue, the genes expressed in callus were distinct from those expressed in other tissues in that a large proportion contained a class 1 CpG island. These results suggest that plant CpG islands may be useful for deducing the expression pattern of uncharacterized genes. [ABSTRACT FROM PUBLISHER]

One of the key end points for understanding the molecular basis of embryogenesis is the analysis of spatiotemporal patterns of gene expression. Methodical limitations due to low mRNA levels often prevent a tissue-specific resolution. In this study, we developed an improved laser microdissection technique and RT-PCR that allows marker gene detection in small tissue areas from sections of formalin-fixed paraffin-embedded Xenopus embryos. Tissue pieces were isolated by laser microbeam microdissection and captured by laser pressure catapulting. Neither laser treatment nor conventional histological or immunochemical staining impaired subsequent RNA analysis. Transcripts of tissue-specific marker genes such as endodermin (endoderm), epidermal cytokeratin (epidermal ectoderm), N-CAM (neural tube), myoD (somites), and sonic hedgehog (floor plate) were amplified by nested RT-PCR analysis from small areas of single sections. [ABSTRACT FROM AUTHOR]

The cloning and sequencing of two bovine connexin32 cDNAs are reported. Comparative analysis with known corresponding mammalian cDNA and protein sequences, besides confirming a high degree of similarity among these proteins, allowed us to identify some specific features of the bovine connexin32 gene. The latter include: the presence of a novel exon in the 5′ UTR which is alternatively spliced, giving rise to a new mRNA species; the presence of two potential hairpin loops in the 5′ and 3′ UTR; and the presence of an additional amino acid, glycine235, in the C‐terminal domain of the 284 residue protein. Among the common features, the presence of polypyrimidine clusters within the 3′ UTR, containing a consensus sequence for a cis‐acting element, is noteworthy. Expression of connexin32 mRNAs was analysed in 16 bovine tissues. Transcript analysis suggests the presence, in cattle, of an alternative downstream promoter. [ABSTRACT FROM AUTHOR]

The cephalochordate amphioxus is thought to share a common ancestor with vertebrates. To investigate the evolution of developmental mechanisms in chordates, cDNA clones for two amphioxus actin genes, BfCA1 and BfMA1, were isolated. BfCA1 encodes a cytoplasmic actin and is expressed in a variety of tissues during embryogenesis, beginning in the dorsolateral mesendoderm of the mid-gastrula. At the open neural plate stage, BfCA1 transcripts accumulate at the bases of the neuroectodermal cells adjacent the presumptive notochord. The 3’ untranslated region of BfCA1 contains a sequence that is similar to the ”zipcode” sequence of chicken β-cytoplasmic actin gene, which is thought to direct intracellular mRNA localization. BfCA1 is also expressed in the notochord through the early larval stage, in the pharynx and in the somites at the onset of muscle-cell differentiation. BfMA1 is a vertebrate-type muscle actin gene, although the deduced amino acid sequence is fairly divergent. Transcripts first appear in the early neurula in the somites as they begin to differentiate into axial muscle cells and persist into the adult stage. In young adults, transcripts are localized in the Z-discs of the muscle cells. Smooth muscle cells around the gill slits and striated muscle cells in the pterygeal muscle also express BfMA1; however, there is never any detectable expression in the notochord, which is a modified striated muscle. Together with the alkali myosin light chain gene AmphiMLC-alk, the sequence and muscle-specific expression of BfMA1 implies a conserved mechanism of muscle cell differentiation between amphioxus and vertebrates. Evolution of the chordate actin gene family is discussed based on molecular phylogenetic analysis and expression patterns of amphioxus actin genes. [ABSTRACT FROM AUTHOR]

The mammalian Nramp1 protein is an integral membrane protein expressed exclusively in macrophages, where it plays a critical role in the ability of these cells to destroy ingested microbes. The bactericidal mechanism of action of Nramp1 remains unknown. We report the identification and characterization of cDNA clones corresponding to three homologues of the mammalian Nramp1 gene from the genome of Oryza sativa, OsNramp1, OsNramp2, and OsNramp3. These three genes encode a novel group of highly similar hydrophobic polypeptides sharing between 64% and 75% sequence similarity, that show similar hydropathy profiles, and predicted secondary structure, including the same number, position, and sequence characteristics (including conserved charges) of transmembrane domains. Together, these define a highly conserved membrane associated hydrophobic core. The three plant proteins show a remarkable degree of sequence similarity with their mammalian counterpart (60% to 70% similarity), including primary and secondary structure elements previously described in ion transporters and channels. Expression studies in normal plant tissues indicate that while OsNramp1 is expressed primarily in roots, and OsNramp2 is primarily expressed in leaves, OsNramp3 is expressed in both tissues. The recent discovery that the yeast Nramp homologue SMF1 functions as a manganese transporter raises the exciting possibility that OsNramp encodes a family of metal ion transporters in plants. [ABSTRACT FROM AUTHOR]

Commercially used natural rubber ( cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree ( Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level. [ABSTRACT FROM AUTHOR]

Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicated a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the N-labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N-sufficient conditions. [ABSTRACT FROM AUTHOR]

Calcium-dependent protein kinases (CDPKs) in plants typically contain a C-terminal calmodulin-like domain with four EF-hand calcium-binding motifs. We have isolated a carrot somatic embryo cDNA clone which encodes a new, divergent isoform of this family, designated CRK (CDPK-related kinase). The catalytic domain of CRK shares a high degree of homology with the catalytic domains of plant CDPKs (53.5% average identity with its two closest phylogenetic relatives, CDPK431 (carrot) and AK1 ( Arabidopsis). However, the C-terminal domain of CRK bears significantly less homology to calmodulin (22.0% identity to barley calmodulin) than other plant CDPKs (38.0% average identity between barley calmodulin and the C-terminal domains of CDPK431 and AK1). This degeneracy also involves the EF-hand motifs of CRK, which have diverged to varying extents. The predicted structure of CRK also contains an extended N-terminal domain 145 amino acids in length possessing a consensus N-myristoylation signal. CRK transcripts are most abundant in somatic embryos, with lesser accumulations in flowers and leaves and lowest levels in roots. Homologous genomic DNA sequences that hybridize with CRK cDNA but not with a carrot CDPK probe have been detected in a variety of higher plant taxa, including monocotyledonous species, suggesting that this CDPK-related kinase is widely conserved among angiosperms. [ABSTRACT FROM AUTHOR]