Heterogenous associations between Gender-Sexuality Alliances and LGBTQ adolescents' maladjustment across individual victimization level.

Gender-Sexuality Alliances (GSAs), which are student-initiated school clubs for LGBTQ youth and allies, can reduce victimization for lesbian, gay, bisexual, transgender, and queer (LGBTQ) youth. This preregistered study identified heterogeneous correlates of GSAs, based on data from an anonymous survey of LGBTQ adolescents aged 13-17 years living in the United States (N = 10,588). In line with the healthy context paradox (Pan et al. [Child Development, 92, 2021, and 1836]), the presence of a GSA exacerbated associations between LGBTQ-based victimization and depressive symptoms, lower self-esteem, and lower academic grades-particularly in transgender youth. Inclusive settings, such as GSAs, might prevent increasing disparities by including tailored strategies to monitor and support more vulnerable, victimized LGBTQ youth.

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform.

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 µL. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

Disruption of pocket protein dream complexes by E7 proteins of different types of human papillomaviruses.

It has been shown that the E7 protein of the high-risk HPV-16 transforms cells in vitro and binds pRB, p107 and p130, so called pocket proteins associated in cells with DREAM proteins, while that of the low-risk HPV-6 does not transform cells and binds p130 but not pRB or p107. These facts may indicate that p130 is essential for the HPV life cycle. To gain further insight into the relationship between HPV E7 proteins and pocket protein-DREAM complexes, E7 proteins of HPVs of various risk categories were expressed via appropriate vectors in T98G cells and the levels of various pocket proteins either total or associated with DREAM were analyzed. The obtained results demonstrated that high-risk HPV-16, HPV-18 and HPV-33, low-risk HPV-1 and HPV-11, and cutaneous HPV-48 disrupted pocket protein-DREAM complexes in T98G cells to a similar extent.

A method for making directed changes to the Fusarium graminearum genome without leaving markers or other extraneous DNA.

A method is described which allows exact targeted changes to the Fusarium graminearum genome, including changes of as little as one particular base pair to gene-size insertions, replacements or modifications. The technique leaves no other DNA in the genome, such as marker genes, and can be used serially to effect multiple complex changes in any desired chromosomal locations. The method is based on our previous finding that after transformation, DNA with homology to F. graminearum DNA recombines itself into the genome in a predictable manner involving multiple tandem copies. We designed a cloning vehicle with a built-in hygromycin-resistance marker (hygB) which can be used to transform the fungus, and with cloning sites to carry DNA with any desired genomic modifications. To effect a desired genomic change the sequence changes of interest are incorporated between two adjacent borders homologous to F. graminearum DNA which will target them to the desired location. This modified DNA is attached within the cloning sites within the vehicle. Transformants are readily obtained in which tandem copies of the vehicle plus insert are inserted between the two genomic border sequence homologues. Progeny of a transformant are subsequently screened for those with a decreased resistance to the antibiotic, and then for those which have completely lost the marker and the entire vehicle, leaving only the desired sequence modifications between the two genomic border sequences which were targeted. This method is demonstrated by exactly replacing the trichodiene synthase (tri5) gene coding sequence (CDS) with that of a green fluorescence protein (gfp) gene with no other genomic changes. This derivative was then re-engineered to replace the gfp CDS with that of the wild type, exactly regenerating the original F. graminearum genome.

Associations of Relationship Experiences, Dating Violence, Sexual Harassment, and Assault With Alcohol Use Among Sexual and Gender Minority Adolescents.

Sexual and gender minority (SGM) adolescents report higher rates of dating violence victimization compared with their heterosexual and cisgender peers. Research on dating violence often neglects diversity in sexual and gender identities and is limited to experiences in relationships. Further, given that dating violence and alcohol use are comorbid, research on experiences of dating violence could provide insights into alcohol use disparities among SGM adolescents. We aimed to map patterns of relationship experiences, sexual and physical dating violence, and sexual and physical assault and explored differences in these experiences among SGM adolescents. Further, we examined how these patterns explained alcohol use. We used a U.S. non-probability national web-based survey administered to 13-17-year-old SGM adolescents (N = 12,534). Using latent class analyses, four patterns were identified: low relationship experience, dating violence and harassment and assault (72.0%), intermediate dating experiences, sexual harassment, and assault and low levels of dating violence (13.1%), high dating experiences, dating violence, and sexual assault (8.6%), and high dating experiences, dating violence, and sexual harassment and assault (6.3%). Compared to lesbian and gay adolescents, bisexual adolescents reported more experiences with dating, dating violence, and sexual assault, whereas heterosexual adolescents reported fewer experiences with dating, dating violence, and sexual harassment and assault. Compared to cisgender boys, cisgender girls, transgender boys, and non-binary/assigned male at birth adolescents were more likely to experience dating violence inside and outside of relationship contexts. Experiences of dating, dating violence, and sexual harassment and assault were associated with both drinking frequency and heavy episodic drinking. Together, the findings emphasize the relevance of relationship experiences when studying dating violence and how dating violence and sexual harassment and assault might explain disparities in alcohol use.

Herpes simplex virus type-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli.

The protein coding region of the herpes simplex virus type-1 glycoprotein D (gD) gene was mapped, and the nucleotide sequence was determined. The predicted amino acid sequence of the gD polypeptide was found to contain a number of features in common with other virus glycoproteins. Insertion of this protein coding region into a bacterial expressor plasmid enabled synthesis in Escherichia coli of an immunoreactive gD-related polypeptide. The potential of this system for preparation of a type-common herpes simplex virus vaccine is discussed.

Targeting an E2F site in the mouse genome prevents promoter silencing in quiescent and post-mitotic cells.

Previous studies have shown that the cell cycle-regulated B-myb promoter contains a conserved E2F binding site that is critical for repressing transcription in quiescent cells. To investigate its significance for permanent promoter silencing, we have inactivated this binding site in the mouse genome. Mice homozygous for the mutant B-mybmE2F allele were fully viable, however, B-myb transcription was derepressed during quiescence in mouse embryo fibroblasts (MEFs) derived from mutant animals. Moreover, it was found that mutation of the E2F site resulted in abnormal maintenance of B-myb expression in senescent MEFs and in differentiated brain tissue. These findings therefore reveal a direct and primary role for repressive E2F complexes in silencing gene expression in post-mitotic cells. Analysis of histone modifications at the promoter showed that histone H3 lysine 9 was constitutively acetylated throughout the cell cycle in homozygous mutant MEFs. This mouse system is the first description of an E2F site mutation in situ and will facilitate the study of E2F function in vivo.

A model for integration of DNA into the genome during transformation of Fusarium graminearum.

Transformants of Fusarium graminearum were derived using linearized DNA of plasmids designed to replace the trichodiene synthase gene, a cutinase gene or a xylanase gene with a hygromycin-resistance marker cassette by homologous recombination between 1-kbp segments of flanking DNA. Most transformants did not exhibit the DNA structure expected of integration by classical double recombination. Instead, they contained linearized plasmid joined end-to-end and variably incorporated into the genome. Transformant types included ectopic integrations and integrations at the target site with or without removal of the targeted gene. We have analyzed a large number of transformants using cloning, PCR and DNA sequencing to determine the structures of their integrated DNA, and describe a model to explain their derivations. The data indicate that 1-3 copies of input DNA are first joined end-to-end to produce either linear or circular structures, probably mediated by the non-homologous end-joining (NHEJ) system. The end-joins typically have 1-5 nucleotides in common and are near or within the original cleavage site of the plasmid. Ectopic integrations occur by attaching linear DNA to two ends of genomic DNA via the same joining mechanism. Integration at the target site is consistent with replication around circularized input DNA, beginning and ending within the flanking homologous DNA, resulting in the integration of multiple copies of the entire structure. This results in deletion or duplication of the target site, or leaves one copy at either end of the integrated multimer. Reiterated DNA in the more complex structures is unstable due to homologous recombination, such that conversion to simpler forms is detected.

Expression of the c-myb and c-myc genes is regulated independently in differentiating mouse erythroleukemia cells by common processes of premature transcription arrest and increased mRNA turnover.

The mechanisms that modulate c-myb mRNA levels in mouse erythroleukemia cells induced toward erythroid differentiation were compared with those that act on c-myc. Both genes exhibited regulation at the levels of premature transcription arrest and RNA turnover. However, these common processes allowed temporally distinct control of gene expression.

Scan-directed mini-incision focused parathyroidectomy: how accurate is accurate enough?

INTRODUCTION Mini-incision focused parathyroidectomy (MI-FP) is advocated as an alternative to bilateral neck exploration (BNE), owing to its reduced morbidity. The site and side of the affected gland is identified preoperatively using a combination of ultrasound and sestamibi scans. However, the acceptable degree of inter-scan concordance required to prompt MI-FP without compromising accuracy is undetermined. METHODS Accuracy of preoperative imaging was determined both individually and in combination for all parathyroidectomies (2007-2014). A grading system (excellent, good, poor) was devised to describe the interscan concordance, which was validated by the operative and histological findings. RESULTS Eighty-nine patients (17 male, 68 female) underwent parathyroidectomy (MI-FP 44, BNE 45). The accuracy of scans interpreted individually was 53% for ultrasound and 60% for sestamibi, with no difference according to surgical technique (P = 0.43, P = 1, respectively). The proportion of interscan concordance was: excellent - 35%, good - 40%, poor 25%. Combined accuracy was 100% for both excellent and good grades but only 13% for those graded poor. Similar rates of normocalcaemia were observed for MI-FP and BNE, while postoperative hypocalcaemia was five times higher in those undergoing BNE. CONCLUSIONS Reduction in the inter-scan concordance from excellent to good does not compromise accuracy. MI-FP could be successfully performed in up to 75% of patients - 25% higher than recommended in national guidelines. Focused parathyroidectomy does not compromise surgical and endocrinological outcomes but boasts a far superior complication rate.

The predictive power of airborne gamma ray survey data on the locations of domestic radon hazards in Norway: A strong case for utilizing airborne data in large-scale radon potential mapping.

It is estimated that exposure to radon in Norwegian dwellings is responsible for as many as 300 deaths a year due to lung cancer. To address this, the authorities in Norway have developed a national action plan that has the aim of reducing exposure to radon in Norway (Norwegian Ministries, 2010). The plan includes further investigation of the relationship between radon hazard and geological conditions, and development of map-based tools for assessing the large spatial variation in radon hazard levels across Norway. The main focus of the present contribution is to describe how we generate map predictions of radon potential (RP), a measure of radon hazard, from available airborne gamma ray spectrometry (AGRS) surveys in Norway, and what impact these map predictions can be expected to have on radon protection work including land-use planning and targeted surveying. We have compiled 11 contiguous AGRS surveys centred on the most populated part of Norway around Oslo to produce an equivalent uranium map measuring 180 km × 102 km that represents the relative concentrations of radon in the near surface of the ground with a spatial resolution in the 100 s of metres. We find that this map of radon in the ground offers a far more detailed and reliable picture of the distribution of radon in the sub-surface than can be deduced from the available digital geology maps. We tested the performances of digital geology and AGRS data as predictors of RP. We find that digital geology explains approximately 40% of the observed variance in ln RP nationally, while the AGRS data in the Oslo area split into 14 bands explains approximately 70% of the variance in the same parameter. We also notice that there are too few indoor data to characterise all geological settings in Norway which leaves areas in the geology-based RP map in the Oslo area, and elsewhere, unclassified. The AGRS RP map is derived from fewer classes, all characterised by more than 30 indoor measurements, and the corresponding RP map of the Oslo area has no unclassified parts. We used statistics of proportions to add 95% confidence limits to estimates of RP on our predictive maps, offering public health strategists an objective measure of uncertainty in the model. The geological and AGRS RP maps were further compared in terms of their performances in correctly classifying local areas known to be radon affected and less affected. Both maps were accurate in their predictions; however the AGRS map out-performed the geology map in its ability to offer confident predictions of RP for all of the local areas tested. We compared the AGRS RP map with the 2015 distribution of population in the Oslo area to determine the likely impact of radon contamination on the population. 11.4% of the population currently reside in the area classified as radon affected. 34% of ground floor living spaces in this affected area are expected to exceed the maximum limit of 200 Bq/m3, while 8.4% of similar spaces outside the affected area exceed this same limit, indicating that the map is very efficient at separating areas with quite different radon contamination profiles. The usefulness of the AGRS RP map in guiding new indoor radon surveys in the Oslo area was also examined. It is shown that indoor measuring programmes targeted on elevated RP areas could be as much as 6 times more efficient at identifying ground floor living spaces above the radon action level compared with surveys based on a random sampling strategy. Also, targeted measuring using the AGRS RP map as a guide makes it practical to search for the worst affected homes in the Oslo area: 10% of the incidences of very high radon contamination in ground floor living spaces (≥800 Bq/m3) are concentrated in just 1.2% of the populated part of the area.

A transcriptional arrest mechanism involved in controlling constitutive levels of mouse c-myb mRNA.

The control of c-myb mRNA abundance was examined in three representative cell lines, (the erythroleukaemia F4-12B2, the myeloma MOPC-31C and the fibroblast NIH3T3), which display abundant, low and undetectable levels of this transcript, respectively. We observed a small difference in half-life between F4-12B2 and MOPC-31C c-myb mRNA (175 min and 105 min, respectively) insufficient to account for the approximately 20-fold lower levels of this transcript in myelomas. Using the run-on transcription assay we found that c-myb transcripts were initiated at similar rates in all three cell types and were elongated at this relatively high rate to a site approximately 2 kilobases into the first intron. NIH3T3 c-myb transcripts did not proceed detectably beyond this pause/attenuation site, while in F4-12B2 cells transcription of regions 3' of this site occurred at a rate approximately 12-fold greater than in MOPC-31C. We have concluded that this transcriptional arrest mechanism, together with small differences in RNA turnover, were sufficient to account for the spectrum of c-myb mRNA abundance observed. Despite evidence of transcript initiation, we were unable to detect c-myb mRNA in fibroblasts, even under conditions (e.g. serum stimulation) which induced high c-myc mRNA levels. However, a novel 3.0 kilobase transcript with homology to c-myb was detected in cycloheximide-treated NIH3T3 cells.

The cell-cycle regulated transcription factor B-Myb is phosphorylated by cyclin A/Cdk2 at sites that enhance its transactivation properties.

Expression of the B-Myb transcription factor is upregulated during late G1 phase of the cell cycle by an E2F-dependent transcriptional mechanism. B-Myb is specifically phosphorylated during S phase, suggesting that a cyclin-dependent kinase (Cdk) regulates its activity. Consistent with this notion, the S phase-specific cyclin A/Cdk2 was found previously to enhance B-Myb transactivation activity in cotransfected cells. In this study we provide evidence that B-Myb is a direct physiological target for cyclin A/Cdk2. We demonstrate that B-Myb is an in vitro substrate for cyclin A/Cdk2, but not for cyclin D1/Cdk4 or cyclin E/Cdk2. By mutating candidate Cdk2 phosphorylation sites, we show that B-Myb is phosphorylated at Thr447, Thr490, Thr497 and Ser581 by cyclin A/Cdk2 in vitro and that these sites are also phosphorylated in cycling U-2 OS cells. Inhibition of endogenous Cdk2 by dominant negative Cdk2 attenuated phosphorylation of Thr447, Thr490 and Thr497, but had no effect upon Ser581 modification. B-Myb transactivation activity was significantly reduced in a mutant containing amino acid substitutions at all four identified cyclin A/Cdk2 sites and was constitutively low in Saos-2 cells where endogenous cyclin A/Cdk2 activity was unable to phosphorylate ectopically expressed B-Myb. These data indicate that phosphorylation by cyclin A/Cdk2 is directly involved in enhancing B-Myb transactivation activity and that levels of endogenous cyclin A/Cdk2 activity may contribute to cell line-specific B-Myb function.

Characterization and cell cycle-regulated expression of mouse B-myb.

We have isolated a full-length mouse B-myb cDNA clone and used this to examine cell cycle-regulated expression of this gene. Mouse B-Myb was predicted to comprise 704 amino acids and to be 84% homologous with human B-Myb. There were three regions of extensive amino acid homology which may indicate functional domains: the first corresponded to the c-Myb DNA-binding domain, while the second had no counterpart in c-Myb but was instead homologous to a short segment of the related A-myb protein. The third region of homology is partially conserved in both c-Myb and A-Myb and may correspond to the c-Myb negative regulatory domain. Stimulation of quiescent 3T3 fibroblasts with serum was found to result in induction of B-myb expression in late G1 and to lead to high levels of gene transcripts that persisted through S phase. Similarly, maximum B-myb mRNA levels were reached in G2/M synchronized cells prior to entry into S phase. These results are consistent with a role in G1/S transition as has been suggested for c-myb.

The induction of Friend erythroleukaemia differentiation is markedly affected by expression of a transfected c-myb cDNA.

A minigene containing a cDNA encoding the normal mouse p80c-myb protein under strong promoter control was used to transfect Friend erythroleukaemia cells. Expression of the transfected gene was found to result in elevated levels of p80c-myb which were not subject to rapid down-modulation by inducers of Friend cell differentiation as was the product of the endogenous c-myb gene. Continued synthesis of p80c-myb was found to be associated with a marked decrease in differentiation of Friend cells and we concluded that normal down-regulation of c-myb expression may be a necessary event in differentiation of these cells.

Inhibition of cyclin A/Cdk2 phosphorylation impairs B-Myb transactivation function without affecting interactions with DNA or the CBP coactivator.

Expression of the B-Myb transcription factor is directed by an E2F-dependent transcriptional mechanism to late G1 and S phases of the cell cycle, where its transactivation properties are enhanced post-translationally by cyclin A/Cdk2-mediated phosphorylation. Other experiments have shown that removal of the B-Myb C-terminus constitutively activates both transactivation and DNA-binding activities, suggesting that autoregulation by this inhibitory domain is counteracted by phosphorylation. We report here on further experiments to examine this hypothesis. The importance of this modification was first emphasized by showing that co-transfected dominant-negative Cdk2 (Cdk2DN) substantially reduced B-Myb transactivation activity. We then attempted to map the autoregulatory domain by analysing a series of progressively deleted C-terminal B-Myb mutants. Removal of just 29 C-terminal aa increased transactivation appreciably, however, maximal activity required removal of 143 amino acids (as in B-Myb + 561). Enhanced B-Myb + 561 function correlated with the acquisition of DNA binding activity to a single Myb binding site (MBS) oligonucleotide as determined by bandshift assays, however, further assays showed that even wt B-Myb could bind a DNA fragment containing three MBS. Although transactivation by B-Myb was severely dependent on hyperphosphorylation, neither inhibiting this activity by co-transfecting Cdk2DN nor augmenting it with cyclin A resulted in significant effects on DNA-binding. We also found that B-Myb could synergize with the CBP coactivator and that this cooperativity was cyclin A/Cdk2-dependent. Despite this, the physical association between these proteins was not influenced by the B-Myb phosphorylation status. We discuss these findings in relation to the autoregulation of B-Myb by the C-terminal domain.

E2F binding is required but not sufficient for repression of B-myb transcription in quiescent fibroblasts.

We have previously shown in mouse NIH3T3 fibroblasts that transcription of the B-myb gene, which encodes a transcription factor required for S phase entry, is repressed through a promoter E2F site in G0/early G1. Transcription repression at this stage of the cell cycle was correlated with binding of a specific p107/E2F complex to this site. We report here, however, that transfection of cells with the known components of this complex, p107, E2F-4 and DP-1, did not repress the B-myb promoter in cycling NIH3T3 cells, although p107 inhibited transcription transactivation by E2F-4/DP-1. To establish definitively the contribution of E2F to repression, the effects of further mutations within and surrounding the E2F site were examined. It was evident that E2F binding and repression were closely correlated, lending greater weight to the contention that E2F itself is implicated in this activity. These studies also identified a closely linked site, designated the downstream repression site (DRS), which was not required for E2F binding or transactivation but which was necessary for repression. These findings indicated that E2F-dependent repression and activation are independently regulated phenomena and suggest that repression involves additional interactions determined by the promoter context.

Rapid dephosphorylation of p107 following UV irradiation.

In response to UV irradiation, mouse NIH3T3 fibroblasts transiently arrest predominantly in the G1 phase of the cell cycle. Here, we investigate the role of the retinoblastoma-related pocket proteins in this biological process. We report here that UV induces an increase in p107/E2F complexes, shown previously to be repressors of E2F-dependent transcriptional activity. Several lines of evidence indicate that the increase of p107/E2F complexes following UV irradiation is a consequence of rapid dephosphorylation of p107. First, UV-mediated p107 dephosphorylation could be abolished by pretreatment of NIH3T3 fibroblasts with the serine/threonine phosphatase inhibitors calyculin A and okadaic acid. Second, alteration of protein phosphatase 2A holoenzyme composition by over-expression of specific B subunits interfered with UV-mediated dephosphorylation of p107. Consistent with this, p107 could be dephosphorylated in vitro with PP2A. Moreover, dephosphorylation of p107 was shown to be independent of the activity of p53 and p21, as it occurred also in UV-treated p53-null as well as p21-null mouse fibroblasts. We observed a close correlation between the UV dosages required for G1 cell cycle arrest and p107 dephosphorylation. Our data suggest a model in which UV radiation-induced cell cycle arrest depends, at least in part, on the induction of a PP2A-like phosphatase that acts on p107.

Analysis of the C4-dicarboxylate transport genes of Rhizobium meliloti: nucleotide sequence and deduced products of dctA, dctB, and dctD.

Rhizobium meliloti transports succinate, fumarate, malate, and aspartate by means of the dicarboxylate transport system, which is encoded by dct genes located on the exo megaplasmid. Analysis of these genes using Tn5 insertion mutagenesis revealed three complementation groups within a 5.9-kb HindIII fragment. The sequence of this fragment and the sites of Tn5 insertion were determined. Three genes, dctA, dctB, and dctD, were identified as the only three open-reading frames in locations consistent with the complementation data. The dctA gene is preceded by the sequence CTGGCACG-N4-TTGCT, which is characteristic of promoters requiring the ntrA-encoded protein for activation. The dctA-encoded protein is highly hydrophobic and contains eight potential transmembrane helices, indicating that it is probably the structural component of the transport system responsible for movement of dicarboxylates from the periplasm across the inner membrane. The dctB and dctD genes are transcribed in the opposite direction to dctA. They encode proteins with homology to the R. leguminosarum bv. viceae dicarboxylate transport proteins regulating expression of dctA and to other proteins comprising two-component regulatory systems. The dctB-encoded protein includes a putative periplasmic N-terminal domain that senses the presence of dicarboxylates and a C-terminal cytoplasmic domain that activates the dctD-encoded protein. The C-terminus of the dctD-encoded protein shows homology to several DNA-binding proteins, indicating that it is probably the domain which binds DNA in the dctA promoter region to regulate dctA transcription. All the R. meliloti mutants altered in dctA, dctB, and dctD were complemented by the dct region from R. l. bv. viceae.

Bacterial synthesis of herpes simplex virus types 1 and 2 glycoprotein D antigens.

We have used elements of the E. coli lactose (lac) operon to produce a collection of herpes simplex virus types 1 and 2 glycoprotein D (gD-1 and gD-2) antigens. Our approach employed recombinant DNA techniques to construct plasmids with various segments of the gD-1 and gD-2 coding sequences fused to the lacZ gene. Such hybrid genes were expressed in a regulated manner in E. coli by joining them to the lac promoter-operator region. Efficient translation of these hybrid genes was facilitated by incorporating a coding sequence specifying a short peptide leader (lambda cro) in the plasmid expression vectors resulting in synthesis of chimeric Cro-gD-beta-galactosidase proteins. In addition, insertion of synthetic translation terminators at the junction of gD and lacZ enabled us to produce specific truncated gD polypeptide sequences unfused to beta-galactosidase. The gD antigens produced in E. coli were not glycosylated and were generally recovered as dense insoluble aggregates. Proteins containing portions of gD-1 or gD-2 were analyzed by immunoprecipitation using anti-HSV rabbit serum and a number of monoclonal antibodies recognizing different epitopes of gD-1. Initial animal studies were done with antigens that reacted with neutralizing antisera or monoclonal antibodies. When these bacterially produced proteins were injected into rabbits, antibodies were produced that specifically immunoprecipitated authentic gD polypeptides and neutralized the infectivity of both virus types. These studies suggest that gene fusion techniques can be used to produce immunogenic proteins in large quantity. These polypeptides are not only useful in analyses of gene structure and function, but also can provide novel diagnostic reagents and well-defined pure antigens for vaccine development.