Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.

A number of the glycoproteins identified on the surfaces of cells of the immune response belong to the immunoglobulin superfamily. We have isolated and characterized cDNA clones and the complete genomic gene encoding a B-cell-specific member of the immunoglobulin superfamily called "B29." This isolate is expressed at all stages in B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy chain gene diversity region

DNA vaccines provide an attractive technology platform against bioterrorism agents due to their safety record in humans and ease of construction, testing, and manufacture. We have designed monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins and tested their immunogenicity and ability to protect rabbits from an aerosolized inhalation spore challenge. Immune responses after two or three injections of cationic lipidformulated PA, PA plus LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA). High titers of anti-PA, anti-LF, and neutralizing antibody to lethal toxin (Letx) were achieved in all rabbits. Eight or nine animals in each group were challenged with 100؋ LD50 of aerosolized anthrax spores 5 or 9 weeks after vaccination. An additional 10 animals vaccinated with PA pDNA were challenged >7 months postvaccination. All animals receiving PA or PA plus LF pDNA vaccines were protected. In addition, 5 of 9 animals receiving LF pDNA survived, and the time to death was significantly delayed in the others. Groups receiving three immunizations with PA or PA plus LF pDNA showed no increase in anti-PA, anti-LF, or Letx neutralizing antibody titers postchallenge, suggesting little or no spore germination. In contrast, titer increases were seen in AVA animals, and in surviving animals vaccinated with LF pDNA alone. Preclinical evaluation of this cationic lipid-formulated bivalent PA and LF vaccine is complete, and the vaccine has received U.S. Food and Drug Administration Investigational New Drug allowance.

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A complete understanding of the factors that determine selection of antigens recognized by the humoral immune response following infectious agent challenge is lacking. Here we illustrate a systems biology approach to identify the antibody signature associated with Brucella melitensis (Bm) infection in humans and predict proteomic features of serodiagnostic antigens. By taking advantage of a full proteome microarray expressing previously cloned 1406 and newly cloned 1640 Bm genes, we were able to identify 122 immunodominant antigens and 33 serodiagnostic antigens. The reactive antigens were then classified according to annotated functional features (COGs), computationally predicted features (e.g., subcellular localization, physical properties), and protein expression estimated by mass spectrometry (MS). Enrichment analyses indicated that membrane association and secretion were significant enriching features of the reactive antigens, as were proteins predicted to have a signal peptide, a single transmembrane domain, and outer membrane or periplasmic location. These features accounted for 67% of the serodiagnostic antigens. An overlay of the seroreactive antigen set with proteomic data sets generated by MS identified an additional 24%, suggesting that protein expression in bacteria is an additional determinant in the induction of Brucella-specific antibodies. This analysis indicates that one-third of the proteome contains enriching features that account for 91% of the antigens recognized, and after B. melitensis infection the immune system develops significant antibody titers against 10% of the proteins with these enriching features. This systems biology approach provides an empirical basis for understanding the breadth and specificity of the immune response to B. melitensis and a new framework for comparing the humoral responses against other microorganisms.

B29 is a B-cell-specific member of the immunoglobulin gene superfamily that is expressed throughout B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy-chain gene segment rearrangements. We have analyzed the region upstream of the B29 gene to identify DNA sequences involved in transcriptional regulation of this gene. The B29 gene lacks a TATA box and transcription is initiated at multiple sites. The B29 gene sequence 5' ofthese transcription start sites contains six promoter and enhancer motifs known to control immunoglobulin gene transcription. The most notable is a perfect octamer (5'-ATTTGCAT-3'), which binds the Oct-2 B-cell-specific transcription factor and thereby can account for the tissue-specific expression of this gene.The cis-acting enhancer and promoter sequence motifs that control immunoglobulin gene transcription have been extensively characterized. Closely related sequences called E motifs were first identified by in vivo footprinting in the heavychain enhancer (1, 2). Three examples of E motifs also are located in the K light-chain enhancer (3). Distinct trans-acting factors have been shown to bind in vitro to the El motif in the heavy-chain enhancer and to the highly similar E3 motifs in the heavy-and light-chain enhancers (3-7). The E motifs show tissue-specific reactivity in vivo (1, 2). However, as assayed in vitro, the factors binding to the E motifs are present in all cells.

c Routine serodiagnosis of herpes simplex virus (HSV) infections is currently performed using recombinant glycoprotein G (gG) antigens from herpes simplex virus 1 (HSV-1) and HSV-2. This is a single-antigen test and has only one diagnostic application. Relatively little is known about HSV antigenicity at the proteome-wide level, and the full potential of mining the antibody repertoire to identify antigens with other useful diagnostic properties and candidate vaccine antigens is yet to be realized. To this end we produced HSV-1 and -2 proteome microarrays in Escherichia coli and probed them against a panel of sera from patients serotyped using commercial gG-1 and gG-2 (gGs for HSV-1 and -2, respectively) enzyme-linked immunosorbent assays. We identified many reactive antigens in both HSV-1 and -2, some of which were type specific (i.e., recognized by HSV-1-or HSV-2-positive donors only) and others of which were nonspecific or cross-reactive (i.e., recognized by both HSV-1-and HSV-2-positive donors). Both membrane and nonmembrane virion proteins were antigenic, although type-specific antigens were enriched for membrane proteins, despite being expressed in E. coli. Herpes simplex virus 1 (HSV-1) and HSV-2 cause significant human morbidity. HSV-2 is the causative agent of most recurrent genital herpes lesions and is sexually transmitted. Infections are often asymptomatic, and most infected individuals are unaware of the infection, yet HSV-2 is associated with an increased risk of HIV acquisition (33) and an increased risk during pregnancy of spontaneous abortion, premature birth, and perinatal herpes (12, 13). Unawareness of HSV-2 infection is also a major contributing factor to transmission to uninfected partners (64,65). In contrast, HSV-1 is usually transmitted during childhood and is found to be associated predominantly with orolabial infections (cold sores). Also, HSV-1 infection of the eye (ocular herpes) is the most common cause of infectious corneal blindness in industrialized countries. Both HSV-1 and HSV-2 establish lifelong latent infections within the dorsal root and trigeminal ganglia and are characterized by periodic reactivation and virus shedding from mucocutaneous epithelium. Owing to the different natural histories and outcomes of HSV-1 and -2 infections, accurate diagnosis of the HSV type is important for patient management and prognosis and controlling potential transmission. For example, knowing the specific HSV type can help the patient take appropriate precautions to prevent transmission of the disease to others. In particular, the identification of unrecognized HSV-2 infection can be used to carefully monitor virus shedding during pregnancy and minimize the risk of perinatal infection.Laboratory tests for HSV infection include virus culture, virus neutralization, PCR, and serological tests. Virus culture is considered the "gold standard" in the early stages of a primary infection. However, it is less sensitive during the healing stage of infection or during recurrent infections. Culture testi...

The localization of mRNAs which encode the glial-specific marker proteins, glial fibrillary acidic protein (GFAP), glycerol phosphate dehydrogenase (GPDH, EC 1.1.1.8), and myelin basic protein (MBP), was mapped by in situ hybridization in primary cultures of 1-2-day-old rat brain in serum-supplemented medium. Developmental changes of these expressed mRNAs were examined after various times in culture ranging from 8 to 50 days and were correlated with the histological, morphological, and positional characteristics of the cells. By day 8, the culture stratified into a population of flat polygonal astrocytes covered by another population of phase-dark process-bearing cells. When counterstained with May-Grunwald histological stain, astrocytes appeared pale blue, whereas two subpopulations of phase-dark cells stained differentially; one was dark blue while the other was red and smaller. GFAP-specific sequences were abundant at day 8, increased in the astrocyte bedlayer as the culture became confluent, and plateaued at approximately day 16. A minor proportion of blue phase-dark cells contained GFAP mRNA although at a lower abundance. In contrast, GPDH mRNA positive blue phase-dark cells were seen scattered throughout the upper layer of the culture and also around the perimeter of large clumps of red phase-dark cells. These cells were infrequent at day 8 but increased in number at later time points. The expression of MBP mRNA differed from GPDH in that it was more abundant at early time points, plateaued between day 20 and day 24, and was predominantly localized in red phase-dark cells.(ABSTRACT TRUNCATED AT 250 WORDS)