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HIV-1 in the developing CNS: developmental differences in gene expression.

HIV-1 infection of the CNS plays a direct role in the pathogenesis of AIDS dementia that frequently accompanies systemic AIDS. Both adult and pediatric AIDS are characterized by a high proportion of CNS disease. However, the pathogenic mechanisms responsible for AIDS dementia are not understood. A transgenic mouse model using the LTRs of two CNS-derived strains of HIV-1 (HIV-1JR-CSF and HIV-1JR-FL) has been developed to study HIV-1 gene expression in vivo. Analyses of expression in adult transgenic mice revealed expression in neurons in the CNS (J. R. Corboy, J. M. Buzy, M. C. Zink, and J. E. Clement, Science 258, 1804-1808, 1992). In this study, developmental analyses of HIV-1-directed gene expression in embryonic and newborn transgenic mice derived from the above lines revealed strikingly different levels and patterns of expression in the CNS and spinal cord compared with adult mice. Increased expression was observed in the newborn brain compared to the adult, and the neuroanatomical pattern of expression was markedly different than that observed in adult brain. Transient expression was detected in the dorsal root ganglia and spinal cord in embryos and newborns up to Day 14. In contrast to the expression in neurons in adult CNS, HIV-1-directed gene expression in the newborn brain was observed in neurons, endothelial cells, and macrophages. This difference in expression during development probably reflects temporally regulated cellular transcription factors in the CNS. This transgenic model suggests that HIV-1 replication in the CNS may use cellular transcription factors different from those in nonneural tissues. Studies are in progress to identify cellular transcription factors that may be responsible for the differential expression of the LTRs.

AIDS Dementia Complex↗

Mitochondrial cytochrome b gene in two developmental stages of human malarial parasite Plasmodium falciparum.

The cytochrome b gene of the mitochondrial ubiquinol-cytochrome c reductase (complex III of electron transport chain) was characterized in two developmental stages of human malarial parasite cultivated in vitro. The cytochrome b gene spanning the nucleotide position 4691 to 5930 in 6-kb mitochondrial DNA from gametocytic (sexual) and intraerythrocytic (asexual) stages of Plasmodium falciparum (a T9,94 mutant line) were in vitro amplified from total DNA using polymerase chain reaction (PCR). It was found that the parasites from both stages contained the PCR product approximately 1.2 kb in length that was localized in mitochondria. The nucleotide sequences of cytochrome b gene at Qi/quinone binding site from both stages were analyzed using thermal cycle sequencing and were found to be the same. The amount of this gene from both stages of the parasite were determined by using the quantitative PCR method. The results showed that the amount of the cytochrome b gene produced from the sexual stage was seven times higher than that obtained from the asexual stage. Our results would provide basic information on the regulation of cytochrome b and the 6-kb mitochondrial DNA during growth and development of the sexual and asexual stages of the malarial parasite in the mammalian host.

Animals↗

Transgenic tobacco over-expressing a homeobox gene shows a developmental interaction between leaf morphogenesis and phyllotaxy.

The tobacco gene, NTH1, encodes a polypeptide of 326 amino acids and is a member of the class1 KN1-type family of homeobox genes. Expression of NTH1 has mainly been observed in vegetative and reproductive shoot apices, not observed in roots or expanded leaves. Over-expression of NTH1 in transgenic plants caused abnormal leaf morphology, consisting of wrinkling and curvature. Interestingly, the direction of leaf curvature tended to be conserved among almost all of the leaves in any given transformant. In transgenic plants exhibiting clockwise or anticlockwise phyllotaxy, leaves curved to the right or left, respectively, when looking from the shoot apex toward the base. Micro-surgical experiments demonstrated that the presence of the shoot apex is necessary for the development of leaf curvature, indicating that the order of formation of leaves on the stem (the generative spiral) affects leaf development. We found a correlation between the severity of leaf curvature and the value of the plastochron ratio, a parameter of phyllotaxy. Transformants with more severe phenotypes had larger plastochron ratios. From these findings, we discuss the possibility that an increase in the plastochron ratio, caused by over-expression of NTH1 in the shoot apex, may be involved in leaf curvature.

Amino Acid Sequence↗

Speciation despite gene flow when developmental pathways evolve.

Evolutionary biologists assume that species formation requires a drastic reduction in gene exchange between populations, but the rate sufficient to prevent speciation is unknown. To study speciation, we use a new class of population genetic models that incorporate simple developmental genetic rules, likely present in all organisms, to construct the phenotype. When we allow replicate populations to evolve in parallel to a new, shared optimal phenotype, often their hybrids acquire poorly regulated phenotypes: Dobzhansky-Muller incompatibilities arise and postzygotic reproductive isolation evolves. Here we show that, although gene exchange does inhibit this process, it is the proportion of migrants exchanged (m) rather than the number of migrants (Nm) that is critical, and rates as high as 16 individuals exchanged per generation still permit the evolution of postzygotic isolation. Stronger directional selection counters the inhibitory effect of gene flow, increasing the speciation probability. We see similar results when populations in a standard two-locus, two-allele Dobzhansky-Muller model are subject to simultaneous directional selection and gene flow. However, in developmental pathway models with more than two loci, gene flow is more able to impede speciation. Genetic incompatibilities arise as frequent by-products of adaptive evolution of traits determined by regulatory pathways, something that does not occur when phenotypes are modeled using the standard, additive genetic framework. Development therefore not only constrains the microevolutionary process, it also facilitates the interactions among genes and gene products that make speciation more likely-even in the face of strong gene flow.

Animals↗

Expression of Na+/Ca(2+) exchanger (NCX1) gene in the developmental mouse embryo and adult mouse brain.

The Na(+)/Ca(2+) exchanger gene, NCX1, is widely expressed in many tissues, encoding several isoforms through alternative RNA splicing. NCX1 deficient mice are known to be lethal at embryonic day 9-10 (E9-10). However, its expression pattern during embryogenesis is largely unknown. Therefore, to identify and compare the localization and alternatively spliced isoforms of NCX1 mRNA expressed in the developmental stages, we analyzed the mouse embryo. Northern blot analysis demonstrated that NCX1 mRNA was expressed from the earliest stage examined, E7. In situ hybridization analysis revealed that NCX1 mRNA was expressed in the heart alone until E10.5. However, at E14.5 and 16.5, NCX1 mRNA was expressed not only in the heart, but also in neuronal cells. In addition, the expression of NCX1 mRNA in the adult brain was most abundant in the hippocampus. Using reverse transcription-polymerase chain reaction (RT-PCR), we also identified the alternatively spliced isoforms expressed during each developmental stage. The restricted expression of the NCX1 gene suggested that NCX1 may play an important role in the developing mouse embryo.

Alternative Splicing↗

Transcriptional potentials of the beta-like globin genes at different developmental stages in transgenic mice and hemoglobin switching.

Developmental-stage-specific regulation and physiological levels of expression of the globin genes can be recaptured in transgenic mice carrying a YAC/BAC- or cosmid-based construct. By contrast, proper developmental regulation and high-level expression cannot be achieved coordinately in transgenic mice carrying a more manipulated construct, such as a plasmid-based globin gene construct. These differences provide us an opportunity to define the requirements for a developmentally regulated, high-level expression of the globin genes in vivo. To achieve this, as a first step, we studied maximum transcriptional potentials of the beta-globin genes at various stages of development. microLCR-enhanced expression of the epsilon-, gamma-, and beta-globin genes driven by their minimal promoters was estimated and compared with that in betaYAC transgenic mice. Quantitative measurements of steady state mRNA levels of the epsilon-, gamma-, and beta-globin genes showed that the microLCR was able to enhance expression of each beta-like globin gene to levels similar to those in the betaYAC mice. Moreover, transcriptional potentials of each globin gene were unchanged during the entire course of development. These observations indicate that the highest level of expression of the globin genes can be achieved in both embryonic and definitive erythropoiesis regardless of developmental specificity of the genes. This finding implies that transcription suppression is the major mechanism of the developmental specificity of the expression of the beta-like globin genes.

Animals↗

A 6-kb upstream region of the human transthyretin gene can direct developmental, tissue-specific, and quantitatively normal expression in transgenic mouse.

To ascertain whether a 6-kb upstream region of the human transthyretin (TTR) gene contains the cis-element(s) required for proper specificity and level of expression, transgenic mice carrying the human mutant TTR gene containing either 6-kb (6.0-hMet30) or 0.6-kb (0.6-hMet30) of the upstream region were produced and studied. The 6.0-hMet30 gene was expressed in the yolk sac, liver, and choroid plexus, where the mouse endogenous TTR gene is also expressed. In contrast, expression of the 0.6-hMet30 gene was restricted to the yolk sac and liver. The expression levels of the 6.0-hMet30 gene in the liver and serum were similar to those of the mouse TTR gene, and about 10-fold those of the 0.6-hMet30 gene. Before birth, the developmental profiles of the expression of both transgenes in each tissue were similar to those of the mouse TTR gene. However, the expression levels of the 6.0-hMet30 gene in the liver and serum increased after birth to reach adult levels at an age of 4 weeks, while expression of the 0.6-hMet30 gene remained at a low level after birth. These results suggest that the 6-kb upstream sequence contains the cis-elements required for developmental, tissue-specific, and quantitatively normal expression.

Animals↗

Differential expression of several E2-type ubiquitin carrier protein genes at different developmental stages in Arabidopsis thaliana and Nicotiana sylvestris.

We characterized three genes encoding different E2-type ubiquitin carrier proteins involved in the ubiquitin-mediated proteolytic pathway: UbcAt3 shows homologies to the yeast CDC34 gene and Ub-cAt4a and UbcAt4b are two different genes homologous to the Ubc1/4/5 subfamily in yeast. Their accumulation was analysed and compared with that of the different families encoding polyubiquitins, as well as the monoubiquitin fusion protein, which is considered as a marker for cell division, during various developmental stages including G0/S transition and senescence of higher plant cells. Our results imply that these Ubc genes are under the control of complex mechanisms, and are differentially regulated, but not necessarily co-regulated with ubiquitin genes. Even the closely related UbcAt4a and UbcAt4b genes of the same multigene subfamily are controlled by distinct regulatory mechanisms.

Adaptation, Physiological↗

Zebrafish cellular nucleic acid-binding protein: gene structure and developmental behaviour.

Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.

Alternative Splicing↗

P63 gene mutations and human developmental syndromes.

The P63 gene is a recently discovered member of the p53 family. While P53 is ubiquitously expressed, p63 is expressed specifically in embryonic ectoderm and in the basal regenerative layers of epithelial tissues in the adult. Complete abrogation of P63 gene function in an animal model points to the relevance of P63 for the proper development of ectodermally derived tissues. The p63 knockout mouse dies at birth and has truncation of the limbs, as well as absence of epidermis, prostate, breast, and urothelial tissues, apparently reflecting ectodermal stem cell loss. A number of dominant human syndromes have been mapped to chromosome 3q27 and ultimately to mutations in the p63 gene. These syndromes have abnormal limb development and/or ectodermal dysplasia and include ectrodactyly, ectodermal dysplasia, clefting syndrome; ankyloblepharon, ectodermal dysplasia, clefting syndrome; acro-dermato-ungual-lacrimal-tooth syndrome; limb-mammary syndrome; as well as nonsyndromic split hand/foot malformation. The pattern of heterozygous mutations is distinct for each of these syndromes. Consistent with this syndrome-specific mutational pattern, the functional consequences of mutations on the p63 proteins also vary, invoking dominant-negative and gain-of-function mechanisms rather than a simple loss of function.

Abnormalities, Multiple↗

The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence.

Aspergillus fumigatus, an important opportunistic pathogen which commonly affects neutropenic patients, produces conidia with a bluish-green color. We identified a gene, alb1, which is required for conidial pigmentation. The alb1 gene encodes a putative polyketide synthase, and disruption of alb1 resulted in an albino conidial phenotype. Expression of alb1 is developmentally regulated, and the 7-kb transcript is detected only during the conidiation stage. The alb1 mutation was found to block 1,3,6,8-tetrahydroxynaphthalene production, indicating that alb1 is involved in dihydroxynaphthalene-melanin biosynthesis. Scanning electron microscopy studies showed that the alb1 disruptant exhibited a smooth conidial surface, whereas complementation of the alb1 deletion restored the echinulate wild-type surface. Disruption of alb1 resulted in a significant increase in C3 binding on conidial surfaces, and the conidia of the alb1 disruptant were ingested by human neutrophils at a higher rate than were those of the wild type. The alb1-complemented strain producing bluish-green conidia exhibited inefficient C3 binding and neutrophil-mediated phagocytosis quantitatively similar to those of the wild type. Importantly, the alb1 disruptant had a statistically significant loss of virulence compared to the wild-type and alb1-complemented strains in a murine model. These results suggest that disruption of alb1 causes pleiotropic effects on conidial morphology and fungal virulence.

Amino Acid Sequence↗

Developmental regulation of genes mediating murine brain glucose uptake.

We examined the molecular mechanisms that mediate the developmental increase in murine whole brain 2-deoxyglucose uptake. Northern and Western blot analyses revealed an age-dependent increase in brain GLUT-1 (endothelial cell and glial) and GLUT-3 (neuronal) membrane-spanning facilitative glucose transporter mRNA and protein concentrations. Nuclear run-on experiments revealed that these developmental changes in GLUT-1 and -3 were regulated posttranscriptionally. In contrast, the mRNA and protein levels of the mitochondrially bound glucose phosphorylating hexokinase I enzyme were unaltered. However, hexokinase I enzyme activity increased in an age-dependent manner suggestive of a posttranslational modification that is necessary for enzymatic activation. Together, the postnatal increase in GLUT-1 and -3 concentrations and hexokinase I enzymatic activity led to a parallel increase in murine brain 2-deoxyglucose uptake. Whereas the molecular mechanisms regulating the increase in the three different gene products may vary, the age-dependent increase of all three constituents appears essential for meeting the increasing demand of the maturing brain to fuel the processes of cellular growth, differentiation, and neurotransmission.

Aging↗

Chromatin structure and the expression of globin-encoding genes.

The developmental regulation of globin gene expression in the chicken has been studied. All of the genes are regulated by a small number of general erythroid factors. In addition, expression of individual members of the family must be controlled in a lineage (stage)-specific manner. In some cases, the relevant factors may be stage specific, but in others they are not confined to one stage, but exert their control through developmentally regulated changes in their abundance within the nucleus. Chromatin structural elements, such as locus control regions and insulators, are also involved in control of eukaryotic gene expression. Because so much is understood about regulation of individual genes, the globin family has proven valuable in investigating control of transcription at the level of chromatin structure.

Animals↗

Gene evolution and regulation in the chorion complex of Bombyx mori. Hybridization and sequence analysis of multiple developmentally middle A/B chorion gene pairs.

Twenty-two pairs of chorion genes belonging to the A and B multigene families have been characterized and mapped within two segments of a 320 kb (1 kb = 10(3) bases or base-pairs) chromosomal walk in the domesticated silkmoth Bombyx mori. Eighteen of the gene pairs belong to two groups that are typified by the previously characterized A/B.L12 and A/B.L11 chorion gene pairs, and are defined by two respective types of short (approx. 280 base-pairs) bidirectional promoter sequences. In the chromosome, the L12-like and L11-like pairs are interspersed with each other and with the remaining four gene pairs, which have unrelated promoter sequences. We have sequenced the promoter regions and adjacent small exons of all L12-like and L11-like A and B genes in the walk. The L12-like promoters are highly conserved, whereas L11-like promoters are somewhat more variable. Reconsideration of previous data on RNA accumulation and disappearance during choriogenesis, in the light of the sequences, indicates that L12-like genes are developmentally early-middle, while L11-like genes correspond to two developmental subgroups, middle I and middle II. Sequence comparisons of all these promoters, as well as the previously characterized promoters of the developmentally late HcA and HcB genes, identify short elements of possible regulatory significance. The sequences, as well as extensive cross-hybridization analysis with short probes derived from the reference A/B.L12 gene pair, under carefully controlled conditions of stringency, indicate the occurrence of sequence transfers among A or B genes. These sequence transfers, which could result from gene conversions or unequal crossovers, are less abundant than in the HcA and HcB families, but do result in a patchwork of similarities and differences in the A and B genes. The transfers appear to be least frequent between the moderately divergent A genes that belong to different temporal classes, while the L12-like and L11-like B genes appear to be extensively homogenized in sequence.

Animals↗

Expression of one of the members of the Arabidopsis chaperonin 60 beta gene family is developmentally regulated and wound-repressible.

To study the pattern of gene regulation of the plastid chaperonin 60 beta gene family a chimaeric gene was constructed fusing the 5'-flanking region of the chaperonin 60 beta B3 gene to the beta-glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.

Arabidopsis↗

DNA sequence analysis of a cyclophilin gene from maize: developmental expression and regulation by salicylic acid.

In plants, such as maize, cyclophilin (Cyp) genes are expressed at a basal level in all tissues. Amounts of Cyp mRNA above the basic level are observed in germinating seedlings, in growing tissues/organs such as roots and leaf meristematic tissue of young maize plants, nodes and embryonic female inflorescences of adult plants and also in non-proliferating tissues such as the internodes of adult plants. Salicylic acid (SA) enhances the transcription of maize Cyp genes. The possible involvement of SA in the pathway leading to defense responses induced by abiotic stresses such as mercuric chloride treatment is discussed. A maize Cyp genomic clone isolated using a maize Cyp cDNA probe contains 737 bp of the 5' upstream and the entire coding region. This Cyp gene is not interrupted by intervening sequences. In the 5' upstream region, characteristic transcription signals as well as putative regulatory sequences were identified. Two TATA boxes are found at positions -56 bp and -66 bp with respect to the transcription start site. Two putative heat shock elements were identified in the promoter region; a metal regulatory element and a third heat shock element were localized in the 5' untranslated leader. Several putative polyadenylation signals and (G)T-rich sequence motifs were identified in the 3' untranslated region.

Amino Acid Isomerases↗

Developmental selection of var gene expression in Plasmodium falciparum.

The protozoan Plasmodium falciparum causes lethal malaria. Adhesion of erythrocytes infected with P. falciparum to vascular endothelium and to uninfected red blood cells (rosetting) may be involved in the pathogenesis of severe malaria. The binding is mediated by the antigenically variant erythrocyte-membrane-protein-1 (PfEMP-1), which is encoded by members of the P. falciparum var gene family. The control of expression and switching of var genes seems to lack resemblance to mechanisms operating in variant gene families of other microbial pathogens. Here we show that multiple, distinct var gene transcripts (about 24 or more) can be detected by reverse transcription and polymerase chain reaction in bulk cultures of the rosetting parasite FCR3S1.2, despite the adhesive homogeneity of the cultures. We also detected several var transcripts in single erythrocytes infected with a ring-stage parasite of FCR3S1.2, and found that different var genes are transcribed simultaneously from several chromosomes in the same cell. In contrast, we detected only one var transcript, FCR3S1.2 var-1, which encodes the rosetting PfEMP-1 protein, in individual rosette-adhesive trophozoite-infected cells, and we found only one PfEMP-1 type at the erythrocyte surface by labelling with 125iodine and immunoprecipitation. We conclude that a single P. falciparum parasite simultaneously transcribes multiple var genes but, through a developmentally regulated process, selects only one PfEMP-1 to reach the surface of the host cell.

Adult↗