BACKGROUND:The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions.RESULTS:Our finishing process was designed to close gaps, improve sequence quality and validate the assembly. Sequence traces derived from the WGS and draft sequencing of individual bacterial artificial chromosomes (BACs) were assembled into BAC-sized segments. These segments were brought to high quality, and then joined to constitute the sequence of each chromosome arm. Overall assembly was verified by comparison to a physical map of fingerprinted BAC clones. In the current version of the 116.9 Mb euchromatic genome, called Release 3, the six euchromatic chromosome arms are represented by 13 scaffolds with a total of 37 sequence gaps. We compared Release 3 to Release 2; in autosomal regions of unique sequence, the error rate of Release 2 was one in 20,000 bp.CONCLUSIONS:The WGS strategy can efficiently produce a high-quality sequence of a metazoan genome while generating the reagents required for sequence finishing. However, the initial method of repeat assembly was flawed. The sequence we report here, Release 3, is a reliable resource for molecular genetic experimentation and computational analysis.

BACKGROUND:A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages.RESULTS:We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40% of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing.CONCLUSIONS:We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5' expressed sequence tags (5' expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ~40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5' ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the ∼120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes ∼13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Vol. 270, p. 12635 We previously reported that an alanine substitution for the serine at position 139 in the I-domain of CD11a did not affect LFA-1 adhesion to ICAM-1. Subsequent to the publication of our paper, we discovered that the S139A construct was actually wild type CD11a. Repeating our adhesion experiments with the appropriate construct demonstrated that mutating serine 139 to alanine completely abrogated LFA-1 binding to ICAM-1, even up to concentrations of 10 μg/ml ICAM-1. The crystal structure of the CD11a I-domain has recently been solved and has shown that serine 139 is involved in coordinating divalent cations. Therefore, our result with the S139A construct is consistent with I-domain cation coordination playing a role in LFA-1/ICAM-1 binding.

Three chimpanzees immunized with recombinant gp120 from human immunodeficiency virus type 1 (HIV-1) strain MN and 1 control animal were challenged intravenously with a primary isolate of HIV-1(SF2). Viral infection was detected in the control animal by viral culture, polymerase chain reaction, and multiple serologic assays beginning 2 weeks after infection, Markers of HIV-1 infection were not detected in any of the gp120-vaccinated animals during 12 months of follow-up, Antisera from the gp120-immunized chimpanzees were unable to neutralize the challenge virus cultured in peripheral blood mononuclear cells (PBMC), These studies demonstrate that immunization with recombinant gp120 derived from a T cell-adapted isolate prevented infection by a heterologous primary isolate of HIV-1. The results suggest that in vitro virus neutralization assays utilizing primary isolates cultured in PBMC may be imperfect indicators of protection in vivo.

We have identified amino acid residues within the evolutionarily conserved I domain of the alpha-chain (CD11a) of the leukocyte integrin leukocyte function-associated antigen (LFA) 1 that are critical for intercellular adhesion molecule (ICAM) 3 (CD50) binding. ICAM-3, a ligand of LFA-1, is thought to mediate intercellular adhesion essential for the initiation of immune responses. Using a panel of human/murine I domain chimeras and point mutants, we observed that the Ile-Lys-Gly-Asn motif, located in the NH2-terminal part of the CD11a I domain, is required for ICAM-3 but not ICAM-1 binding. These findings demonstrate that the I domain of CD11a contains distinct functional subdomains for ligand specific binding. An aspartic acid located at position 137, which is essential to ICAM-1/LFA-1 interactions (Edwards, C.P., M. Champe, T. Gonzalez, M.E. Wessinger, S.A. Spencer, L.G. Presta, P.W. Berman, and S.C. Bodary. 1995. J. Biol. Chem. 270:12635-12640), was also critical for ICAM-3 binding, whereas Ser at position 139 did not effect ICAM-1 or ICAM-3 binding. A synthetic peptide containing the Ile-Lys-Gly-Asn motif inhibited ICAM-3-dependent adhesion and proliferation of T cells at micromolar concentrations, suggesting that this peptide interferes with immune recognition. These observations underscore the importance of ICAM-3 in leukocyte function, and may lead to development of a new category of immunosuppressive agents.

Lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is involved in leukocyte adhesion during cellular interactions essential for immunologic responses and inflammation. mAbs against LFA-1 have been shown to inhibit several T cell-dependent immune functions in vitro and prevent graft failure after bone marrow transplantation in vivo. A murine anti-human CD11a mAb, MHM24, has been humanized and shown to prevent adhesion of human T cells to human keratinocytes and the proliferation of T cells in response to nonautologous leukocytes in the mixed lymphocyte response assay. However, of the nonhuman primate CD11a that we tested, the murine and humanized mAbs cross-reacted only with chimpanzee CD11a. To have a mAb available for preclinical studies in rhesus monkeys, the humanized mAb was reengineered to bind to rhesus CD11a by changing four residues in one of the complementarity-determining regions, CDR-H2, in the variable heavy domain. Cloning and molecular modeling of rhesus CD11a I-domain suggested that the changes from Lys197 and/or Arg189 in human CD11a I-domain to Glu197 and Gln189 in rhesus CD11a I-domain may be the reason that rhesus CD11a does not bind to the murine and humanized mAbs.

Several studies indicate that the I domain located in the alpha chain (CD11a) of leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) plays an essential role in ligand recognition. me recently identified three distinct epitopes (IdeA, IdeB, and IdeC) within the CD11a I domain, recognized by antibodies that block binding of LFA-1 to intercellular adhesion molecules (ICAM) 1, 2, and 3. In the present study, we used a series of human/ murine CD11a I domain chimeras, to localize a fourth I domain epitope (IdeD), recognized by three independently derived anti-CD11a antibodies that selectively block the binding of LFA-1 to ICAM-3, but not to ICAM-1. The IdeD epitope depended on human CD11a residues Asp(182) and Ser(184) and was not present in CD11b or CD11c. Although mutation of Asp(182) and Ser(184) failed to abolish ICAM-3 adhesion of LFA-1 transfectants, alignment of these residues with the crystal structure of the CD11a I domain suggested that the IdeD epitope is located in close proximity to residues (Ile(126) and Asn(129)) recently implicated in the ICAM-3 binding site (1). Interestingly, the IdeB and IdeC epitopes appeared to be in close proximity of a divalent cation binding pocket within the CD11a I domain that regulates both ICAM-1 and ICAM-3 adhesion. Taken together, these data indicate that distinct regions of the CD11a I domain contain epitopes for antibodies that either selectively inhibit binding of LFA-1 to ICAM-3, or interfere with both ICAM-1 and ICAM-3 binding of LFA-1.

Leukocyte function-associated antigen-1 (LFA-1) is a cell surface adhesion receptor for intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, -3). Using human/murine chimeras of the I domain of the LFA-1 alpha subunit (CD11a), we recently identified the epitopes recognized by eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the entire human I domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross conformation or heterodimer formation of LFA-1, as assayed by antibody binding, In order to assess which residues of the I-domain are responsible for binding to ICAM-1, we tested the ability of a panel of human/murine I-domain chimeras to bind to human ICAM-1. When complexed with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which differ in human and murine I domains may not play a critical role in LFA-1 binding to ICAM-1. A series of point mutations of residues that are conserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated, Substitution of alanine for proline at position 192 in the human CD11a I-domain abrogated adhesion of LFA-1 to ICAM-1. Antibody binding data suggested that this was due to conformational changes within the I domain. Mutation of the aspartic acids at positions 137 and 239 to either alanine or lysine completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids are required for binding of human LFA-1 to human ICAM-1.

The epitopes recognized by eight independently isolated monoclonal antibodies to the alpha chain of human and murine leukocyte function-associated antigen 1 (LFA-1), all able to inhibit receptor function, were identified. Initial localization of epitopes was accomplished using chimeric proteins constructed by splicing fragments of cDNAs encoding the alpha subunit of LFA-1 (CD11a) and the alpha subunit of the closely related leukocyte integrin, Mac-1 (CD11b). Antibody binding to CD11a/CD11b chimeras, expressed in the 293 human kidney cell line, demonstrated that the epitopes recognized by six monoclonal antibodies to human CD11a were located in a similar to 200-amino acid sequence found in all beta(2)-integrin alpha subunits, termed the inserted (I) domain. Three distinct epitopes within the I domain (IdeA, IdeB, and IdeC) were identified using a series of mutants in which sequences from murine CD11a were substituted into human CD11a. A series of mutants incorporating single amino acid substitutions was used to identify individual amino acids essential for antibody binding. The location of these residues accounts for the binding specificity of LFA-1-blocking antibodies and identifies particular conserved sequences (residues 126-150) in the I domain of CD11a and homologous sequences in other beta(2)-integrin alpha subunits that may be important for ligand binding.

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC50) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.

Vaccines prepared from the envelope glycoprotein, gp120, of the common laboratory isolate of human immunodeficiency virus type 1 (HIV-1) (IIIB/LAV-1) elicit antibodies that neutralize the homologous virus but show little if any cross-neutralizing activity. This may be because the principal neutralizing determinant (PND) of gp120 is highly unusual in the IIIB/LAV-1 strain and is not representative of those found in the majority of field isolates. We have now examined the immunogenicity of recombinant gp120 prepared from the MN strain of HIV-1 (MN-rgp120), whose PND is thought to be representative of approximately 60% of the isolates in North America. Our results show that MN-rgp120 is a potent immunogen and elicits anti-gp120 titers comparable to those found in HIV-1-infected individuals. While both MN-rgp120 and IIIB-rgp120 induced antibodies able to block gp120 binding to CD4, strain-specific and type-common blocking antibodies were detected. Finally, antibodies to MN-rgp120 but not to IIIB-rgp120 were effective in neutralizing a broad range of laboratory and clinical isolates of HIV-1. These studies demonstrate that susceptibility or resistance to neutralization by antibodies to gp120 correlates with the PND sequence and suggest that the problem of antigenic variation may not be insurmountable in the development of an effective AIDS vaccine.