Molecular Cloning of Vacuolar H+-Pyrophosphatase and Its Developmental Expression in Growing Hypocotyl of Mung Bean1

Vacuolar proton-translocating inorganic pyrophosphatase and H+-ATPase acidify the vacuoles and power the vacuolar secondary active transport systems in plants. Developmental changes in the transcription of the pyrophosphatase in growing hypocotyls of mung bean (Vigna radiata) were investigated. The cDNA clone for the mung bean enzyme contains an uninterrupted open reading frame of 2298 bp, coding for a polypeptide of 766 amino acids. Hypocotyls were divided into elongating and mature regions. RNA analysis revealed that the transcript level of the pyrophosphatase was high in the elongating region of the 3-d-old hypocotyl but was extremely low in the mature region of the 5-d-old hypocotyl. The level of transcript of the 68-kD subunit of H+-ATPase also decreased after cell maturation. In the elongating region, the proton-pumping activity of pyrophosphatase on the basis of membrane protein was 3 times higher than that of H+-ATPase. After cell maturation, the pyrophosphatase activity decreased to 30% of that in the elongating region. The decline in the pyrophosphatase activity was in parallel with a decrease in the enzyme protein content. These findings indicate that the level of the pyrophosphatase, a main vacuolar proton pump in growing cells, is negatively regulated after cell maturation at the transcriptional level.

Developmental abnormalities and epimutations associated with DNA hypomethylation mutations.

A number of aberrant morphological phenotypes were noted during propagation of the Arabidopsis thaliana DNA hypomethylation mutant, ddm1, by repeated self-pollination. Onset of a spectrum of morphological abnormalities, including defects in leaf structure, flowering time, and flower structure, was strictly associated with the ddm1 mutations. The morphological phenotypes arose at a high frequency in selfed ddm1 mutant lines and some phenotypes became progressively more severe in advancing generations. The transmission of two common morphological trait syndromes in genetic crosses demonstrated that the phenotypes are caused by heritable lesions that develop in ddm1 mutant backgrounds. Loss of cytosine methylation in specific genomic sequences during the selfing regime was noted in the ddm1 mutants. Potential mechanisms for formation of the lesions underlying the morphological abnormalities are discussed.

A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3- cells to colonize lymph nodes.

IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions.

Histone H1, a major structural component of chromatin fiber, is believed to act as a general repressor of transcription. To investigate in vivo the role of this protein in transcription regulation during development of a multicellular organism, we made transgenic tobacco plants that overexpress the gene for Arabidopsis histone H1. In all plants that overexpressed H1 the total H1-to-DNA ratio in chromatin increased 2.3-2.8 times compared with the physiological level. This was accompanied by 50-100% decrease of native tobacco H1. The phenotypic changes in H1-overexpressing plants ranged from mild to severe perturbations in morphological appearance and flowering. No correlation was observed between the extent of phenotypic change and the variation in the amount of overexpressed H1 or the presence or absence of the native tobacco H1. However, the severe phenotypic changes were correlated with early occurrence during plant growth of cells with abnormally heterochromatinized nuclei. Such cells occurred considerably later in plants with milder changes. Surprisingly, the ability of cells with highly heterochromatinized nuclei to fulfill basic physiological functions, including differentiation, was not markedly hampered. The results support the suggestion that chromatin structural changes dependent on H1 stoichiometry and on the profile of major H1 variants have limited regulatory effect on the activity of genes that control basal cellular functions. However, the H1-mediated chromatin changes can be of much greater importance for the regulation of genes involved in control of specific developmental programs.

Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects.

The essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear ribonucleoprotein to the branch site. Human U2AF (hU2AF) is a heterodimer composed of a large (hU2AF65) and small (hU2AF35) subunit. Although these proteins associate in a tight complex, the biochemical requirement for U2AF activity can be satisfied solely by the large subunit. The requirement for the small subunit in splicing has remained enigmatic. No biochemical activity has been found for hU2AF35 and it has been implicated in splicing only indirectly by its interaction with known splicing factors. In the absence of a biochemical assay, we have taken a genetic approach to investigate the function of the small subunit in the fruit fly Drosophila melanogaster. A cDNA clone encoding the small subunit of Drosophila U2AF (dU2AF38) has been isolated and sequenced. The dU2AF38 protein is highly homologous to hU2AF35 containing a conserved central arginine- and serine-rich (RS) domain. A recessive P-element insertion mutation affecting dU2AF38 causes a reduction in viability and fertility and morphological bristle defects. Consistent with a general role in splicing, a null allele of dU2AF38 is fully penetrant recessive lethal, like null alleles of the Drosophila U2AF large subunit.

Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.

In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis.

Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.

Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.

Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and developmental success.

This study describes a paternal effect on sperm aster size and microtubule organization during bovine fertilization. Immunocytochemistry using tubulin antibodies quantitated with confocal microscopy was used to measure the diameter of the sperm aster and assign a score (0-3) based on the degree of radial organization (0, least organized; 3, most organized). Three bulls (A-C) were chosen based on varying fertility (A, lowest fertility; C, highest fertility) as assessed by nonreturn to estrus after artificial insemination and in vitro embryonic development to the blastocyst stage. The results indicate a statistically significant bull-dependent difference in diameter of the sperm aster and in the organization of the sperm astral microtubules. Insemination from bull A resulted in an average sperm aster diameter of 101.4 microm (76.3% of oocyte diameter). This significantly differs (P < or = 0.0001) from the average sperm aster diameters produced after inseminations from bull B (78.2 microm; 60.8%) or bull C (77.9 microm; 57.8%), which themselves displayed no significant differences. The degree of radial organization of the sperm aster was also bull-dependent. Sperm asters organized by bull A-derived sperm had an average quality score of 1.8, which was higher than that of bull B (1.4; P < or = 0.0005) or bull C (1.2; P < or = 0.0001). Results with bulls B and C were also significantly different (P < or = 0.025). These results indicate that the paternally derived portion of the centrosome varies among males and that this variation affects male fertility, the outcome of early development, and, therefore, reproductive success.

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.

A developmental timer regulates degradation of cyclin E1 at the midblastula transition during Xenopus embryogenesis.

We have analyzed cyclin E1, a protein that is essential for the G1/S transition, during early development in Xenopus embryos. Cyclin E1 was found to be abundant in eggs, and after fertilization, until the midblastula transition (MBT) when levels of cyclin E1 protein, and associated kinase activity, were found to decline precipitously. Our results suggest that the reduced level of the cyclin E1 protein detected after the MBT does not occur indirectly as a result of degradation of the maternally encoded cyclin E1 mRNA. Instead, the stability of cyclin E1 protein appears to play a major role in reduction of cyclin E1 levels at this time. Cyclin E1 protein was found to be stable during the cleavage divisions but degraded with a much shorter half-life after the MBT. Activation of cyclin E1 protein turnover occurs independent of cell cycle progression, does not require ongoing protein synthesis, and is not triggered as a result of the ratio of nuclei to cytoplasm in embryonic cells that initiates the MBT. We therefore propose that a developmental timing mechanism measures an approximately 5-hr time period, from the time of fertilization, and then allows activation of a protein degradative pathway that regulates cyclin E1. Characterization of the timer suggests that it might be held inactive in eggs by a mitogen-activated protein kinase signal transduction pathway.

UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression.

The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression. It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes. This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression. After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci. Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels. In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced. The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1. These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes.

The new dysmorphology: application of insights from basic developmental biology to the understanding of human birth defects.

Information obtained from studies of developmental and cellular processes in lower organisms is beginning to make significant contributions to the understanding of the pathogenesis of human birth defects, and it is now becoming possible to treat birth defects as inborn errors of development. Mutations in genes for transcription factors, receptors, cell adhesion molecules, intercellular junctions, molecules involved in signal transduction, growth factors, structural proteins, enzymes, and transporters have been identified in genetically caused human malformations and dysplasias. The identification of these mutations and the analysis of their developmental effects have been greatly facilitated by the existence of natural or engineered models in the mouse and even of related mutations in Drosophila, and in some instances a remarkable conservation of function in development has been observed, even between widely separated species.