Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the a, I, and intercalary heterochromatic regions. Screening of a Agtll cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a AgtlO library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.In eucaryotic organisms, the genetic material exists as a complex between DNA, histones, and nonhistone chromosomal (NHC) proteins. The histones, whose primary structure has been highly conserved over evolution, are the protein components of the nucleosomes and as such are associated with almost all the DNA sequences of the genome. NHC proteins, therefore, are the leading candidates to play specific roles in the organization of higherorder chromatin structure and in the control of gene expression. It appears very likely that specific NHC proteins are involved in the compaction of nucleosomes into domains, the formation of specialized structures such as centromeres and telomeres, and the condensation of the chromatin fiber into the mitotic chromosome structure (9, 12). The presence or absence of various NHC proteins may also be critical in establishing the differences in structure between euchromatin and heterochromatin in the interphase nucleus.While a great deal of success has been obtained in the characterization of those NHC proteins with a known enzymatic function (16, 24), a systematic biochemical approach to the study of other NHC proteins has proven to be a difficult task. This may be attributed in part to their unusual physico-chemical properties (25), relatively low abundance, and lack of any functional assays. We chose an approach that allowed us to identify NHC proteins of Drosophila melanogaster by what is essentially a structural assay. Monoclonal antibodies prepared against fractionated nuclear proteins of D. melanogaster embryos are used in immunofluorescence staining of the polytene chromosomes of thirdinstar larvae of D. melanogaster. Those monoclonal antibodies that indicate a protein distrib...

We report here that a point mutation in the gene which encodes the heterochromatin-specific nonhistone chromosomal protein HP-I in Drosophila melanogaster is associated with dominant suppression of position-effect variegation. The mutation, a G-to-A transition at the first nucleotide of the last intron, causes missplicing of the HP-1 mRNA. This suggests that heterochromatin-specific proteins play a central role in the gene suppression associated with heterochromatic position effects.The partitioning of eukaryotic chromosomes into regions which differ in their degrees of compaction has long been appreciated. Most of the transcriptionally active chromatin appears to decondense after mitotic telophase into euchromatin, but a substantial fraction of chromosomal material remains condensed as heterochromatin. Heterochromatin replicates relatively late in the cell cycle and, in tissues which undergo polytenization, the heterochromatin may be underreplicated.The potential of heterochromatin formation to result in transcriptional inactivation is inferred from two genetic phenomena: Barr-body formation (Lyonization) in mammalian females and position-effect variegation in a variety of organisms (reviewed in ref. 1). In both cases chromosomal regions which are euchromatic under some circumstances assume the morphology of heterochromatin. The condensed structure observed in these cases is strongly correlated with transcriptional inactivity.In Drosophila, the genetic dissection of heterochromatin is aided by the availability of numerous rearrangements which lead to variegated expression of euchromatic genes that have come to be relocated near the heterochromatic breakpoint. A number of loci have been identified which, when mutated, act as dominant modifiers of such variegating position effects (2-7). Many of these loci are believed to encode chromatin proteins or factors that modify chromatin structure (see refs. 8 and 9 for recent reviews).A heterochromatin-specific chromosomal protein called HP-1 has been identified and characterized in D. melanogaster (10, 11). A cDNA encoding this protein has been cloned (10), and the gene has been localized to cytological position 29A on the polytene chromosome map. In this report, we provide the sequence of the gene$, identifying exon and intron boundaries, and present molecular evidence that a point mutation at one boundary, causing missplicing of the HP-1 pre-mRNA, is associated with dominant suppression of heterochromatic position effect. This indicates a requirement for HP-1 protein in generating normal heterochromatin structure. MATERIALS AND METHODSDrosophila Stocks. Su(var)205/In(2LR)CyO and the iso-2nd line (marked with b It rl) were obtained from T. Grigliatti (University of British Columbia, Vancouver). Flies were cultured in half-pint plastic bottles at room temperature, using a cornmeal-based medium supplemented with dried bakers' yeast.Northern Blot Analysis. Total nucleic acids were purified from several flies essentially according to the method of Meyerowitz and H...

Antimicrobial peptides (AMPs) are essential components of innate immunity in a range of species fromDrosophila 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 proteinspredicted 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.

Modifiers of position-effect-variegation in Drosophila encode proteins that are thought to modify chromatin, rendering it heritably changed in its expressibility. In an attempt to identify similar modifier genes in other species we have utilized a known sequence homology, termed chromo box, between a suppressor of position-effect-variegation, Heterochromatin protein 1 (HP1), and a repressor of homeotic genes, Polycomb (Pc). A PCR generated probe encompassing the HP1 chromo box was used to clone full-length murine cDNAs that contain conserved chromo box motifs. Sequence comparisons, in situ hybridization experiments, and RNA Northern blot analysis suggest that the murine and human sequences presented in this report are homologues of the Drosophila HP1 gene. Chromo box sequences can also be detected in other animal species, and in plants, predicting a strongly conserved structural role for the peptide encoded by this sequence. We propose that epigenetic (yet heritable) changes in gene expressibility, characteristic of chromosomal imprinting phenomena, can largely be explained by the action of such modifier genes. The evolutionary conservation of the chromo box motif now enables the isolation and study of putative modifier genes in those animal and plant species where chromosomal imprinting has been described.