Mapping of a mouse homolog of a heterochromatin protein gene the X chromosome.

Modifiers of position-effect-variegation in Drosophila are thought to encode proteins that are either structural components of heterochromatin or enzymes that modify these components. We have recently shown that a sequence motif found in one Drosophila modifier gene, Heterochromatin protein 1 (HP1), is conserved in a wide variety of animal and plant species (Singh et al. 1991). Using this motif, termed chromo box, we have cloned a mouse candidate modifier gene, M31, that also shows considerable sequence homology to Drosophila HP1. Here we report evidence of at least four independently segregating loci in the mouse homologous to the M31 cDNA. One of these loci--Cbx-rs1--maps to the X Chromosome (Chr), 1 cM proximal to Amg and outside the X-inactivation center region.     

The Identification and Localization of a Human Gene with Sequence Similarity toPolycomblikeofDrosophila melanogaster

TheDrosophilaPolycomb group (PcG) of genes is required for the epigenetic regulation of a number of important developmental genes, including the homeotic (Hox) genes. The members of this gene family encode proteins that do not share sequence similarity, implying that each plays a unique role in this epigenetic repression mechanism.Polycomblike(Pcl) was the second PcG gene to be identified. We report here the isolation and characterization of a human cDNA, termedPHF1,which encodes a protein with significant sequence similarity toDrosophilaPolycomblike (PCL). The region of similarity between PHF1 and PCL includes the two PHD fingers (C₄–H–C₃motif), the region between them, and sequences C-terminal to the PHD fingers. PHF1 and PCL are 34% identical over this 258-residue region.PHF1was mapped to 6p21.3 by fluorescencein situhybridization. While several genetic diseases that are likely to result from developmental abnormalities map to this region,PHF1is not a clear candidate gene for any of them.     

A Novel Zinc Finger-Containing RNA-Binding Protein Conserved from Fruitflies to Humans

TheDrosophila larkgene encodes an essential RNA-binding protein of the RNA recognition motif (RRM) class that is required during embryonic development. Genetic analysis demonstrates that it also functions as a molecular element of a circadian clock output pathway, mediating the temporal regulation of adult emergence in the fruitfly. We now report the molecular characterization of a human gene with significant similarity tolark.Based on fluorescencein situhybridization and radiation hybrid mapping, the human gene has been localized to chromosome region 11q13; it is closely linked to several identified genes including the locus of Bardet–Biedl syndrome type 1. Thelark-homologous human gene expresses a single 1.8-kb size class of mRNA in most or all tissues including brain. Additional database searches have identified a mouse counterpart that is virtually identical to the human protein. Similar to lark protein, both mammalian proteins contain two copies of the RRM-type consensus RNA-binding motif. Unlike most RRM family members, however, theDrosophilaand mammalian proteins also contain a retroviral-type (RT) zinc finger that is situated 43 residues C-terminal to the second RRM element. Within a 184-residue segment spanning the RRM elements and the RT zinc finger, the human and mouse proteins are 61% similar to theDrosophilalark sequence. These common sequence features and comparisons among a large collection of RRM proteins suggest that the human and mouse proteins represent homologues ofDrosophilalark.     

The mechanism of telomere protection: a comparison between Drosophila and humans

Drosophila telomeres are maintained by transposition of specialized retrotransposons rather than by telomerase activity, and their stability is independent of the sequence of DNA termini. Recent studies have identified several proteins that protect Drosophila telomeres from fusion events. These proteins include the telomere capping factors HP1/ORC-associated protein (HOAP) and heterochromatin protein 1 (HP1), the Rad50 and Mre11 DNA repair proteins that are required for HOAP and HP1 localization at telomeres, and the ATM kinase. Another telomere-protecting factor identified in Drosophila is UbcD1, a polypeptide highly homologous to class I ubiquitin-conjugating E2 enzymes. In addition, it has been shown that HP1 and both components of the Drosophila Ku70/80 heterodimer act as negative regulators of telomere length. Except for HOAP, all these proteins are conserved in humans and are associated with human telomeres. Collectively, these results indicate that Drosophila is an excellent model system for the analysis of the mechanisms of telomere maintenance. In past and current studies, 15 Drosophila genes have been identified that prevent telomeric fusion, and it has been estimated that the Drosophila genome contains at least 40 genes required for telomere protection. We believe that the molecular characterization of these genes will lead to identification of many novel human genes with roles in telomere maintenance.     

Gene CG17604 of Drosophila melanogaster May Be a Functional Homolog of Yeast Gene ZIP1 and Mammal Gene SCP1 (SYCP1) Encoding Proteins of the Synaptonemal Complex

From data on the molecular organization of transverse filament proteins of the synaptonemal complex (SC)—Zip1 in yeast and SCP1 in mammals—and on the width of the SC central space in these organisms and in Drosophila, the putative molecular structure and size of a transverse filament protein of the SC in Drosophila has been inferred. Using genetic and molecular databases and software from the Internet, we carried out in silico screening for a candidate gene for the Drosophila transverse filament protein. As a most likely candidate, gene c(3)G was chosen. The search in the 250-kb region overlapping the locus of this gene (sections 88E-89B) and containing 78 predicted genes has revealed only one gene,CG17604, whose protein meets all requirements for the transverse filament protein of the SC. It was suggested that gene CG17604is gene c(3)G. In this case, genec(3)G must be localized in section 89A7-8 of the cytological map of Drosophila melanogaster.     

Genetic screening for modifiers of the DREF pathway in Drosophila melanogaster: identification and characterization of HP6 as a novel target of DREF

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. By genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express DREF in the eye discs, we identified 24 genes that suppressed and 12 genes that enhanced the rough eye phenotype when heterozygous for mutations. Five genes, HP6, pigeon, lace, X box binding protein 1 and guftagu were found to carry replication-related element (DRE) sequences in their 5′-flanking regions. Of these, the HP6 gene carries two sequences that match seven out of eight nucleotides of DRE and two additional sequences that match six out of eight nucleotides of DRE in the 5′-flanking region. Band mobility shift assays using Drosophila Kc cell nuclear extracts demonstrated DREF binding to two of these sites and chromatin immunoprecipitation using anti-DREF antibodies confirmed that this occurs in vivo. Knockdown of DREF in Drosophila S2 cells decreased the HP6 mRNA level. The results, taken together, indicate that DREF directly regulates expression of the HP6 gene. HP6 mRNA was detected throughout development by RT-PCR with highest levels in adult males. In addition, immunostaining analyses revealed colocalization of HP6 and DREF in nuclei at the apical tips in the testes.     

Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken

Antimicrobial peptides (AMPs) are essential components of innate immunity in a range of species from Drosophila to humans and are generally thought to act by disrupting the membrane integrity of microbes. In order to discover novel AMPs in the chicken, we have implemented a bioinformatic approach that involves the clustering of more than 420,000 chicken expressed sequence tags (ESTs). Similarity searching of proteins—predicted to be encoded by these EST clusters—for homology to known AMPs has resulted in the in silico identification of full-length sequences for seven novel gallinacins (Gal-4 to Gal-10), a novel cathelicidin and a novel liver-expressed antimicrobial peptide 2 (LEAP-2) in the chicken. Differential gene expression of these novel genes has been demonstrated across a panel of chicken tissues. An evolutionary analysis of the gallinacin family has detected sites—primarily in the mature AMP—that are under positive selection in these molecules. The functional implications of these results are discussed.     

Active MHC class Ib genes in rat are pseudogenes in the mouse

The most telomeric class I region of the MHC in rat and mouse is the M region, which contains about 20 class I genes or gene fragments. The central part carries three class I genes—M4, M5, and M6—which are orthologous between the two species. M4 and M6 are pseudogenes in the mouse but transcribed, intact genes in the rat. To analyze the pseudogene status for the mouse genes in more detail, we have sequenced the respective exons in multiple representative haplotypes. The stop codons are conserved in all mouse strains analyzed, and, consistent with the pseudogene status, all strains show additional insertions and deletions, taking the genes further away from functionality. Thus, M4 and M6 indeed have a split status. They are silent in the mouse but intact in the closely related rodent, the rat.GenBank accession numbers: AF057065 to AF057072 (exon 3 of H2-M4 of reported mouse strains), AF057976 to AF057985 (exon 3 of RT1.M4 of reported rat strains), AF058923 and AF058924 (exon 2 of RT1.M4 of strains PVG and BN), AY286080 to AY286092 (exon 4 of H2-M6 of reported mouse stains), and AY303772 (full-length genomic sequence of RT1.M6-1^l)     

A High-Resolution Genetic Map of the Nervous Locus on Mouse Chromosome 8

The nervous (nr) mutant mouse displays two gross recessive traits: both an exaggeration of juvenile hyperactivity and a pronounced ataxia become apparent during the third and fourth postnatal weeks. Using an intersubspecific intercross, we have established a high-resolution map of a segment of mouse Chromosome 8 that places thenrlocus in a genomic segment defined byD8Rck1on the centromeric end andD8Mit3on the telomeric end. This map position places thenrlocus within the BALB/cGr congenic region of the C3HeB/FeJ-nrstrain, confirming the accuracy of our study. We used this map position to identify and evaluate three genes—ankyrin 1, cortexin, and farnesyltransferase—as candidates for thenrgene. These three genes were eliminated from consideration but allowed us to establish the conservation of synteny between the region containing thenrlocus and a segment of the short arm of human chromosome 8 (8p21–p11.2). Finally, the incomplete penetrance of thenrphenotype led us to perform a screen for modifier loci, and we present evidence that such a nervous modifier locus may exist on mouse Chromosome 5.     

Tctex3, related to Drosophila Polycomblike, is expressed in male germ cells and mapped to the mouse t-complex

Tctex3 showing restricted expression in male germ cells has been isolated during the process of chromosome walking in the mouse t-complex region. The total sequence of Tctex3 cDNA predicts a protein of 580 amino acids with two C4HC3 type PHD fingers. The region containing this conserved motif is shared among members of the Polycomblike proteins that include the mouse M96 and Drosophila Polycomblike. A partial cDNA for a human homolog of Tctex3, HUTEX3, has also been isolated. Mouse Tctex3 gene was mapped adjacent to Tsc2 gene on mouse Chromosome (Chr) 17, and HUTEX3 was located closely to HSET gene in the HLA class II region of chromosome 6. Received: 10 April 1998 / Accepted: 22 June 1998     

Chromosomal distribution of heterochromatin protein 1 (HP1) in Drosophila: a cytological map of euchromatic HP1 binding sites

The Heterochromatin Protein 1 (HP1) is a conserved protein which is best known for its strong association with the heterochromatin of Drosophila melanogaster. We previously demonstrated that another important property of HP1 is its localization to the telomeres of Drosophila, a feature that reflects its critical function as a telomere capping protein. Here we report our analysis of the euchromatic sites to which HP1 localizes. Using an anti-HP1 antibody, we compared immunostaining patterns on polytene chromosomes of the Ore-R wild type laboratory strain and four different natural populations. HP1 was found to accumulate at specific euchromatic sites, with a subset of the sites conserved among strains. These sites do not appear to be defined by an enrichment of known repetitive DNAs. Comparisons of HP1 patterns among several Drosophila species revealed that association with specific euchromatic regions, heterochromatin and telomeres is a conserved characteristic of HP1. Based on these results, we argue that HP1 serves a broader function than typically postulated. In addition to its role in heterochromatin assembly and telomere stability, we propose that HP1 plays an important role in regulating the expression of many different euchromatic regions.     

SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing

This review summarizes genetic, molecular and biochemical studies of the SU(VAR)3-9 protein and the evidence for its key role in heterochromatin formation and heterochromatic gene silencing. The Su(var)3-9 locus was first identified as a dominant modifier of position-effect variegation (PEV) in Drosophila melanogaster. Together with Su(var)2-5 and Su(var)3-7, Su(var)3-9 belongs to the group of haplo-suppressor loci which show a triplo-dependent enhancer effect. All three genes encode heterochromatin-associated proteins. Su(var)3-9 is epistatic to the PEV modifier effects of Su(var)2-5 and Su(var)3-7, and it also dominates the effect of the Y chromosome on PEV. These genetic data support a central role of the SU(VAR)3-9 protein in heterochromatic gene silencing, one that is correlated with its activity as a histone H3-K9 methyltransferase (HMTase). In fact, SU(VAR)3-9 is the main chromocenter-specific HMTase of Drosophila. SU(VAR)3-9 and HP1, the product of Su(var)2-5, are main constituents of heterochromatin protein complexes and the interaction between these two proteins is interdependent. Functional analysis in fission yeast, Drosophila and mammals demonstrate that SU(VAR)3-9-dependent gene silencing processes are conserved in these organisms. This is also demonstrated by the rescue of Drosophila Su(var)3-9 mutant phenotypes with human SUV39H1 transgenes.     

Genetic control of IgM responses to (T,G)-A — L

The primary antibody response to aqueous immunization with a low molecular-weight lot of (T,G)-A — L (#420) was studied in congenic pairs of inbred mouse strains. Two new genetic controls were identified, both of which quantitatively regulate the production of IgM anti-(T,G)-A — L antibody. Testing of F₁ and F₂ progeny demonstrated that one of these genes is linked to the major histocompatibility (H-2) complex, and that high response is dominant over low response. Whether this gene is identical toIr-1A is not yet known. The other gene, designatedIg-TGAL _M , is linked to the immunoglobulin heavy-chain allotype locus (Ig-1) and is expressed in a genedose dependent manner. Following secondary challenge with (T,G)-A — L 420, quantitative differences in IgG antibody response were observed inIr-1A high-responder congenics differing only at theIg-1 locus. Breeding studies, however, failed to demonstrate any linkage between this locus and the quantitative control of IgG anti-(T,G)-A — L antibody. These data demonstrate thatH-2-linked immune response genes can regulate IgM as well as IgG antibody responses, that genetic control of the IgM response to (T,G)-A — L is linked toIg-1, and that bothH-2-linked andIg-1-linked genes may simultaneously affect an IgM antibody response to the same antigen.Abbreviations used in this paper are (T,G)-A — L poly-l-(Tyr,Glu)-poly-d,l-Ala-poly-l-Lys - NMS normal mouse serum - SRBC sheep red blood cells - i.p. intraperitoneal - PBS phosphate-buffered saline - RAMG polyvalent rabbit anti-mouse globulin - 2-Me 2-Mercaptoethanol - 2-MeS 2-Me-sensitive - PFC plaque-forming cells - ABC antigen-binding cells     

Yeast Srp1, a nuclear protein related toDrosophila and mouse pendulin, is required for normal migration, division, and integrity of nuclei during mitosis

This paper describes genes from yeast and mouse with significant sequence similarities to aDrosophila gene that encodes the blood cell tumor suppressor pendulin. The protein encoded by the yeast gene, Srp1p, and mouse pendulin share 42% and 51% amino acid identity withDrosophila pendulin, respectively. All three proteins consist of 10.5 degenerate tandem repeats of 42 amino acids each. Similar repeats occur in a superfamily of proteins that includes theDrosophila Armadillo protein. All three proteins contain a consensus sequence for a bipartite nuclear localization signal (NLS) in the N-terminal domain, which is not part of the repeat structure. Confocal microscopic analysis of yeast cells stained with antibodies against Srp1p reveals that this protein is intranuclear throughout the cell cycle. Targeted gene disruption shows thatSRP1 is an essential gene. Despite their sequence similarities,Drosophila and mouse pendulin are unable to rescue the lethality of anSRP1 disruption. We demonstrate that yeast cells depleted of Srp1p arrest in mitosis with a G2 content of DNA. Arrested cells display abnormal structures and orientations of the mitotic spindles, aberrant segregation of the chromatin and the nuclei, and threads of chromatin emanating from the bulk of nuclear DNA. This phenotype suggests that Srplp is required for the normal function of microtubules and the spindle pole bodies, as well as for nuclear integrity. We suggest that Srp1p interacts with multiple components of the cell nucleus that are required for mitosis and discuss its functional similarities to, and differences fromDrosophila pendulin.     

HP1/ORC complex and heterochromatin assembly

We have used the highly conserved heterochromatin component, heterochromatin protein 1 (HP1), as a molecular tag for purifying other protein components of Drosophila heterochromatin. A complex of HP1 associated with the origin recognition complex (ORC) and an HP1/ORC-associated protein (HOAP) was purified from the maternally loaded cytoplasm of early Drosophila embryo. We propose that the DNA-binding activities of ORC and HOAP function to recruit underphosphorylated isoforms of HP1 to sites of heterochromatin nucleation. The roles of highly phosphorylated HP1, other DNA-binding proteins known to interact with HP1, and histone modifying activities in heterochromatin assembly are also addressed.