Functional antagonism between members of the myb family: B-myb inhibits v-myb-induced gene activation.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that control the expression of certain target genes in immature hematopoietic cells. Here, we report the isolation of a myb-related chicken gene, chicken B-myb. We show that expression of B-myb, unlike that of c-myb, is not restricted to hematopoietic cells, suggesting that B-myb functions in a broader spectrum of cell types than c-myb. We have identified the authentic chicken B-myb protein as a nuclear protein of approximately 110 kDa. We show that the B-myb protein specifically recognizes v-myb binding sites in vitro and that binding is mediated by an N-terminally located DNA binding domain. Although B-myb protein recognizes myb binding sites, B-myb fails to transactivate several myb-responsive gene constructs as well as the endogenous myb-responsive gene mim-1. Instead, we find that B-myb represses v-myb- and c-myb-mediated activation of the mim-1 gene, most likely by competing with other myb proteins for binding sites. Our results raise the possibility that B-myb is an inhibitory member of the myb family.     

Characterization of the gene conversions between the multigene family members of the yeast genome

Stanley Sawyer's gene conversion detection method, implemented in his GENECONV computer program, was used to detect and characterize the gene conversions between the multigene family members of the yeast genome. This method gave different gene conversion frequencies and size distribution for gene families with two members and multigene families with more than two members. The 69 gene conversions detected in multigene families with more than two members occur at a frequency of 7.8% gene conversion/pair of genes compared and have an average size of 173+/-220 nucleotides. Larger gene conversions are found only between more similar genes, the genes involved in gene conversions are distributed almost randomly among the 16 yeast chromosomes, and the frequency of gene conversions increases as the distance between repeated genes decreases. In contrast to previous studies, no relationship was observed between the level of expression of a gene and its involvement in gene conversions. These analyses also suggest that gene conversions might occur by different mechanisms in closely linked genes and unlinked genes. The excess of converted regions at the 3? end of unlinked genes suggests that recombination with incomplete cDNA molecules is the main mechanism responsible for gene conversions between such genes.     

Characterization of zebrafish PSD-95 gene family members

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism.     

Novel roles for APC family members and Wingless/Wnt signaling during Drosophila brain development

Construction of the brain is one of the most complex developmental challenges. Wnt signals shape all tissues, including the brain, and the tumor suppressor adenomatous polyposis coli (APC) is a key negative regulator of Wnt/Wingless (Wg) signaling. We carried out the first assessment of the role of APC proteins in brain development, simultaneously inactivating both APC1 and APC2 in clones of cells in the Drosophila larval optic lobe. We focused on the medulla, where epithelial neural progenitors shift from symmetric to asymmetric divisions across the lateral-medial axis. Loss of both APCs triggers dramatic defects in optic lobe development. Double mutant cells segregate from wild-type neighbors, while double mutant neurons form tangled axonal knots, suggesting changes in cell adhesion. Strikingly, phenotypes are graded along the anterior-posterior axis. Activation of Wg signaling downstream of APC mimics these phenotypes, a dominant-negative TCF blocks them, and a known Wg target, decapentaplegic, is activated in double mutant clones, strongly suggesting that the phenotypes result from activated Wg signaling. We also explored the roles of classic cadherins in differential adhesion. Finally, we propose a model suggesting that Wg signaling regulates fine scale cell fates along the anterior-posterior axis, in part by creating an adhesion gradient and consider possible alternate explanations for our observations.     

The Drosophila retained/dead ringer gene and ARID gene family function during development

The recently discovered ARID family of proteins interact with DNA through a phylogenetically conserved sequence termed the A/T Interaction Domain (ARID). The retained/dead ringer (retn/dri) gene of Drosophila melanogaster is a founding member of the ARID gene family, and of the eARID subfamily. This subfamily exhibits an extended region of sequence similarity beyond the core ARID motif and a separate conserved domain termed the REKLES domain. retn/dri is involved in a range of developmental processes, including axis patterning and muscle development. The retn/dri ARID motif has been shown by in vitro studies to exhibit sequence-specific DNA binding activity. Here we demonstrate that the ARID domain is essential for the in vivo function of retn/dri during embryonic development by showing that a mutant form of RETN/DRI, deleted for part of the ARID domain and unable to bind DNA in vitro, cannot rescue the retn/dri mutant phenotype. In the presence of wild-type RETN/DRI this construct acts as a dominant negative, providing additional support for the proposal that RETN/DRI acts in a multiprotein complex. In contrast, we are yet to find an in vivo role for the REKLES domain, despite its clear evolutionary conservation. Finally, we have used germline clone analysis to reveal a requirement for retn/dri in the Drosophila preblastoderm syncytial mitoses.     

Functional characterization of the Arabidopsis ubiquitin-specific protease gene family reveals specific role and redundancy of individual members in development

Ubiquitin-specific proteases (UBPs) are a highly conserved family of proteins in eukaryotes, and play critical roles in protein de-ubiquitination. Here we report a systematic genetic and expression profiling analysis of the UBP gene family in the Arabidopsis thaliana genome. Mutation analysis of 25 of the 27 member genes representing 13 of the 14 sub-families of the UBP gene family revealed that single-gene mutants of three genes in two sub-families exhibit visible phenotypes. Two of these three genes belonging to the UBP15 sub-family were selected for further characterization. The ubp15 mutants display narrower, serrated and flat rosette leaves, partially due to a defect in cell proliferation, as well as other phenotypes such as early flowering, weak apical dominance and reduced fertility, while the line over-expressing UBP15 shows opposite phenotypes. We demonstrated that UPB15 has UBP activity in vitro , and that this biochemical activity is essential for its in vivo function. A genetic interaction analysis among members of this sub-family revealed that UBP15 and UBP16, but not UBP17, have functional redundancy. Our data thus suggest that distinct UBPs, even within a closely related sub-family, can function in different developmental pathways. Although there are clearly functional redundancies among related sub-family members, those redundancies cannot be inferred simply based on the amino acid identity of the family members.     

Characterization of zebrafish PSD‐95 gene family members

The PSD‐95 family of membrane‐ associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD‐95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD‐95, PSD‐93, and two isoforms of SAP‐97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan‐MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD‐95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Identification of novel members reveals the structural and functional divergence of lepidopteran-specific Lipoprotein_11 family

30K proteins (30KPs) are classified into the lepidopteran-specific Lipoprotein_11 family. They are involved in various physiological processes such as energy storage, embryonic development, and immune response in the silkworm. To date, 30KPs were only found in Bombyx mori and Manduca sexta. Moreover, the C-termini of ENF peptide binding proteins (ENF-BPs) show similarity to 30KPs. ENF peptides are multifunctional insect cytokines and involved in growth regulation and defense reaction, whereas ENF-BPs act as active regulators of ENF peptides. In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived genome and EST datasets. We identified 73 30KP homologous genes in 12 lepidopteran species, of which 56 are novel members. The structural and phylogenetic analyses revealed that these genes could be classified into three groups: ENF-BP genes, typical 30KP genes, and serine/threonine-rich 30KP (S/T-rich 30KP) genes. The C-terminal regions are common to all the three subfamilies, but the N-termini are highly variable. We found a novel subfamily of Lipoprotein_11 and named it S/T-rich 30KP according to its exclusive S/T-rich domain in the N terminus. ENF-BP was also found to contain a special domain in the N terminus, which is homologous to Pp-0912 of Pseudomonas putida. Microarray data and semi-quantitative RT-PCR showed that the three groups have their respective temporal–spatial expression patterns. S/T-rich 30KP genes have enriched expression in the mature testis and might be involved in spermiogenesis or fertilization. Typical 30KP genes are expressed mainly in the fat body and integument at the larvae and pupae stages. ENF-BP genes are expressed predominantly in the hemocyte. The differential spatial–temporal expression profiles revealed the functional divergence of three Lipoprotein_11 subfamilies.     

Role of BMP family members during kidney development.

Members of the Bone morphogenetic protein (BMP) family have been shown to be important signaling molecules throughout mouse development. Accordingly, many BMPs are also expressed during organogenesis of the metanephric kidney. However, only BMP7 has been shown to be absolutely required for proper formation of the kidney, thus the majority of information known involves this family member. BMP7 is expressed in both the ureteric epithelium and the mesenchyme throughout embryonic development and has been shown to function as a survival factor for the nephrogenic mesenchyme. However, there has been some controversy over the role of BMP7 as an inducing molecule for the metanephric mesenchyme. Recent studies have shown that BMP7 functions as an anti-differentiation factor for this mesenchyme cell population. The function of BMPs in the ureter and in the more differentiated epithelial structures of the nephron is less well understood.     

Characterization of five members of the actin gene family in the sea urchin

Hybridization of an actin cDNA clone (pSA38) to restriction enzyme digests of Strongylocentrotus purpuratus DNA indicates that the sea urchin genome contains at least five different actin genes. A sea urchin genomic clone library was screened for recombinants which hydridize to pSA38 and four genomic clones were isolated. Restriction maps were generated which indicate that three of these recombinants contain different actin genes, and that the fourth may be an allele to one of these. The restriction maps suggest that one clone contains two linked actin genes. This fact, which was confirmed by heteroduplex analysis, indicates that the actin gene family may be clustered. The linked genes are oriented in the same direction and spaced about 8.0 kilobases apart. In heteroduplexes between genomic clones two intervening sequences were seen. Significant homology is confined to the actin coding region and does not include any flanking sequence. Southern blot analysis reveals that repetitive DNA sequences are found in the region of the actin genes.     

Pax基因家族在果蝇发育过程中的调控作用

Pax是一个在进化上相当保守的基因家族, 它们编码的产物是一组极为重要的转录调控因子, 并存在于从果蝇到人类的各种生物体中, 参与细胞内信号传导通路的调控, 在胚胎发育过程中对细胞分化、更新、凋亡起重要的调控作用, 影响器官和组织的形成。果蝇中已发现10个Pax基因家族成员, 它们对果蝇胚胎发育及成虫组织器官的分化有非常重要的调控作用。文章结合最新的研究进展, 就果蝇中Pax基因的结构、表达模式和主要功能做一简要综述。     

Detecting and characterizing gene conversions between multigene family members

We used a variety of methods to detect known gene conversions in the actin gene families of five angiosperm species, the beta-globin gene families of two primate species, and the Zfx/Zfy gene families of seven mammalian species. Our goal was to devise a working strategy which would allow the analysis of the members of a multigene family in order to determine whether there had been gene conversions between its members, identify the genes involved in the gene conversions, establish the lengths of the converted regions, and determine the polarities of the gene conversions. We show that three phylogenetic methods and the homoplasy test of Maynard Smith and Smith perform relatively poorly on our data sets because the sequences we analyzed had large levels of multiple substitutions. The method of Sawyer, the compatibility method of Jakobsen and Easteal, the partition matrix method of Jakobsen, Wilson, and Easteal, and the co-double method of Balding, Nichols, and Hunt can be used to identify the genes which have been involved in gene conversions. The co-double method is more powerful than other methods but requires orthologous sequences from related species. Compatibility, phylogenetic, and nucleotide substitution distribution statistics methods can be used to identify the location of the converted region(s). Site-by-site compatibility analyses can also be used to identify the direction of the conversion event(s). Combinations of these methods can therefore be used to establish the presence, locations, and polarities of gene conversions between multigene family members.