Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae.

We have cloned a functional centromeric DNA sequence from Saccharomyces cerevisiae. Using the 2 mu chromosome-loss mapping technique and meiotic tetrad analysis, we have identified this DNA sequence as the centromere of chromosome V (CEN5). The CEN5 sequence has been localized on an 1,100-base-pair BamHI-BglII restriction fragment. Plasmids containing CEN5 and an autonomously replicating sequence are mitotically stable in S. cerevisiae and segregate in a Mendelian fashion during meiosis.

Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells.

We previously reported the isolation of yeast mutants that seem to affect the function of certain autonomously replicating sequences (ARSs). These mutants are known as mcm for their defect in the maintenance of minichromosomes. We have now characterized in more detail one ARS-specific mutation, mcm1-1. This Mcm1 mutant has a second phenotype; MAT alpha mcm1-1 strains are sterile. MCM1 is non-allelic to other known alpha-specific sterile mutations and, unlike most genes required for mating, it is essential for growth. The alpha-specific sterile phenotype of the mcm1-1 mutant is manifested by its failure to produce a normal amount of the mating pheromone, alpha-factor. In addition, transcripts of the MF alpha 1 and STE3 genes, which encode the alpha-factor precursor and the alpha-factor receptor, respectively, are greatly reduced in this mutant. These and other properties of the mcm1-1 mutant suggest that the MCM1 protein may act as a transcriptional activator of alpha-specific genes. We have cloned, mapped and sequenced the wild-type and mutant alleles of MCM1, which is located on the right arm of chromosome XIII near LYS7. The MCM1 gene product is a protein of 286 amino acid residues and contains an unusual region in which 19 out of 20 residues are either aspartic or glutamic acid, followed by a series of glutamine tracts. MCM1 has striking homology to ARG80, a regulatory gene of the arginine metabolic pathway located about 700 base-pairs upstream from MCM1. A substitution of leucine for proline at amino acid position 97, immediately preceding the polyanionic region, was shown to be responsible for both the alpha-specific sterile and minichromosome-maintenance defective phenotypes of the mcm1-1 mutant.

Mutants of S. cerevisiae defective in the maintenance of minichromosomes.

We have isolated yeast mutants that are defective in the maintenance of circular minichromosomes. The minichromosomes are mitotically stable plasmids, each of which contains a different ARS (autonomously replicating sequence), a centrometeric sequence, CEN5, and two yeast genes, LEU2 and URA3. Forty minichromosome maintenance-defective (Mcm-) mutants were characterized. They constitute 16 complementation groups. These mutants can be divided into two classes, specific and nonspecific, by their differential ability to maintain minichromosomes with different ARSs. The specific class of mutants is defective only in the maintenance of minichromosomes that carry a particular group of ARSs irrespective of the centromeric sequence present. The nonspecific class of mutants is defective in the maintenance of all minichromosomes tested irrespective of the ARS or centromeric sequence present. The specific class may include mutants that do not initiate DNA replication effectively at specific ARSs present on the minichromosomes; the nonspecific class may include mutants that are affected in the segregation and/or replication of circular plasmids in general.

Clinical evaluation of a new recombinant antigen-based cytomegalovirus immunoglobulin M immunoassay in liver transplant recipients.

BACKGROUND: Human cytomegalovirus (CMV) is a significant cause of morbidity and mortality among transplant recipients. Monitoring transplant recipients by CMV IgM serology has been questioned by several studies due to the reported insensitivity of serologic tests relative to antigen detection methods. METHODS: In this retrospective study, we have evaluated the performance of the new recombinant antigen-based Abbott AxSYM CMV IgM assay and compared it with CMV culture technique in a cohort of 40 liver transplant recipients who did not receive antiviral prophylaxis. RESULTS: The sensitivity, specificity, and positive and negative predictive values for detection of CMV disease by the AxSYM CMV IgM assay were 90.0%, 60.0%, 69.2%, and 85.7%, respectively, and by culture the values were 100%, 55.0%, 69.0%, and 100%, respectively. Detection of CMV IgM occurred before or at the time of CMV disease in only R+ recipients. CONCLUSION: Although this assay is a sensitive test for CMV-specific IgM, detection of CMV IgM preceded detection of virus by culture in patients only when the liver transplant recipient was CMV immune before transplantation (R+).

Quantitative immunolocalization of a P29 protein (GRA7), a new antigen of toxoplasma gondii.

Ultrastructural localization of a P29 protein of Toxoplasma gondii was examined on thin sections by an immunogold technique using a P29 antigen-specific monoclonal antibody (5-241-178). Immunolocalization of the P29 protein in extracellular tachyzoites demonstrated that this antigen was present in the dense granules. Thus, we have identified this P29 antigen as the seventh protein (GRA7) to be localized to the dense granules of T. gondii. P29 immunolocalization in intracellular tachyzoites demonstrated association of this antigen with the parasite membrane complex, tubular elements of the intravacuolar network, and with the parasitophorous vacuolar membrane. Our immunolabeling data suggest trafficking of the P29 (GRA7) antigen from the dense granule via the intravacuolar network to the parasitophorous vacuolar membrane on invasion of the tachyzoite into the host cell. (J Histochem Cytochem 46:1411-1421, 1998)

New developments in the diagnosis of maternal and congenital CMV infection.

Recent advances in the screening of pregnant women with Cytomegalovirus (CMV) IgM, CMV IgG and CMV IgG avidity serologic tests, has led to a more accurate diagnosis of CMV infection. When serologic screening is performed early in gestation, it is possible to identify those women at risk of intrauterine transmission of the virus, i.e., those women with a primary CMV infection, who should be enrolled in prenatal diagnosis. The use of quantitative PCR on amniotic fluid from pregnant women at 21-22 weeks of gestation in prenatal diagnosis is an effective diagnostic tool to distinguish between CMV infection and CMV disease in the fetus and newborn. Quantitative PCR on peripheral blood leukocytes from CMV infected newborns can be used to monitor viral load, especially during treatment with ganciclovir. These advances in serology and quantitative virology should lead to more accurate diagnosis of maternal and congenital CMV infection.

Diagnosis of and screening for cytomegalovirus infection in pregnant women.

No single diagnostic test for cytomegalovirus (CMV) infection is currently available for pregnant women at all stages of gestation. Improved accuracy in estimating the timing of primary infections can be used to identify women at higher risk of giving birth to congenitally infected infants. A diagnostic algorithm utilizing immunoglobulin G (IgG), IgM, and IgG avidity was used to prospectively screen serum from 600 pregnant women enrolled from two groups: < or =20 weeks gestation (n = 396) or >20 weeks gestation (n = 204). PCR testing of urine and/or blood was performed on all seropositive women (n = 341). The majority (56.8%) of women were CMV IgG seropositive, with 5.5% being also CMV IgM positive. In the IgM-positive women, 1.2% had a low-avidity IgG, indicating a primary CMV infection and a high risk of intrauterine transmission. Two infants with asymptomatic CMV infection were born of mothers who had seroconverted in the second trimester of pregnancy. Baseline, age-stratified CMV serostatus was established from 1,018 blood donors. Baseline seropositivity from a blood donor population increased with age from 34.9% seroprevalence at less than 20 years of age to 72% seroprevalence at 50 years of age. Women at high risk of intrauterine transmission of CMV were identified at all stages of gestation. Women infected with CMV during late gestation may be more likely to transmit the virus, so failure to detect seroconversions in late gestation may result in failure to detect infected neonates.

Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay.

Serological detection of human cytomegalovirus (HCMV)-specific antibody varies greatly because of antigen composition and the lack of antigen standardization. Antigenic materials composed of single well-characterized viral proteins or portions of them, produced via molecular biology, have proven to be promising tools in improving serodiagnosis. We constructed a recombinant protein containing two regions of ppUL32 (p150) and half of ppUL44 (p52) and compared the immunoglobulin M (IgM) reactivity of this triple-antigen fusion protein with that of a double-antigen fusion protein containing the two ppUL32 fragments and that of a monoantigen fusion protein containing half of ppUL44. We also constructed and tested two other monoantigen fusion proteins containing a large fraction of ppUL80a and a fraction of ppUL83. More than 700 serum samples from different groups of immunocompetent and immunosuppressed subjects were tested for the presence of HCMV IgM by recombinant enzyme immunoassay (rec-EIA) and by a commercially available EIA. Western blotting (immunoblotting) and (in the case of immunosuppressed individuals) antigenemia tests by immunofluorescence and PCR of polymorphonuclear leukocytes were also carried out. The results obtained demonstrate that (i) the triple-antigen fusion protein can replace the individual proteins; (ii) the triple-antigen fusion protein cannot be used alone to replace the virus or infected cells in the serological detection of anti-CMV IgM; (iii) the addition of the fusion proteins containing portions of ppUL83 and ppUL80a is essential for the formation of an antigenic mixture that can replace the virus for the search of HCMV-specific IgM; (iv) rec-EIA is very specific and is more sensitive than the commercially available EIA, and the results obtained are consistent with those obtained by Western blotting; and (v) rec-EIA can reliably be used to detect HCMV-specific IgM in different groups of patients with active HCMV infection.

Search for cytomegalovirus-specific immunoglobulin M: comparison between a new western blot, conventional western blot, and nine commercially available assays.

We tested 101 serum samples obtained from pregnant women for the presence of human cytomegalovirus (HCMV)-specific IgM with nine different commercially available kits and with two Western blotting (WB) tests previously developed in our laboratory. The conventional WB test contains viral structural proteins separated on a gel from purified HCMV particles and transferred to nitrocellulose. The new WB test contains viral structural proteins and three recombinant proteins which contain the significant immunogenic portions of pp150 (ppUL32), pp52 (ppUL44), and p130 (pUL57). The results obtained indicate that the new WB test combines high specificity (92.5%) with high sensitivity (95.0%), characteristics that, in combination, have not been obtained with any of the other tests.

A novel Western blot test containing both viral and recombinant proteins for anticytomegalovirus immunoglobulin M detection.

We devised a novel Western blot (WB) test for anti-human cytomegalovirus (HCMV) immunoglobulin M (IgM) detection which contains viral structural polypeptides, significant portions of recombinant p150 (ppUL32), and a significant portion of the most immunogenic nonstructural protein p52 (ppUL44). This new test was evaluated in latently infected blood donors, pregnant women, and transplant recipients with ongoing HCMV infection and shown to be more sensitive and specific than traditional WB and conventional enzyme immunoassay for the detection of HCMV-specific IgM.

The DNA-binding protein pUL57 of human cytomegalovirus: comparison of specific immunoglobulin M (IgM) reactivity with IgM reactivity to other major target antigens.

In this work we used PCR to amplify the DNA regions coding for two polypeptides from pUL57 of human cytomegalovirus (amino acids 540 to 601 and 1144 to 1233) and showed that both portions reacted very efficiently with immunoglobulin M in sera of acutely infected subjects. However, pUL57 is not an essential antigen for the replacement of or supplement to a cocktail of recombinant protein antigens containing portions of ppUL32, -44, -83, and -80a in immunoglobulin M serology.

Detection of serum immunoglobulin M to human cytomegalovirus by western blotting correlates better with virological data than detection by conventional enzyme immunoassay.

Western blotting (immunoblotting) with proteins separated from purified human cytomegalovirus (HCMV) particles (viral WB) has repeatedly been shown to be a reliable and sensitive method for detecting HCMV-specific immunoglobulin M (IgM). The aim of the present work was to determine whether IgM detected by viral WB correlates with virological diagnosis better than conventional enzyme immunoassay (conv-EIA). The presence of an active HCMV infection was documented on the basis of isolation of virus from urine and/or saliva and on the basis of antigenemia and/or PCR with polymorphonuclear leukocytes for immunocompetent and immunocompromised subjects, respectively. The agreement observed between IgM detected by viral WB and the results obtained by virological detection of HCMV was significantly higher (88.7%) than the agreement of IgM detected by conv-EIA and virological results (67.5%).

Organization of a cluster of erythromycin genes in Saccharopolyspora erythraea.

We used a series of gene disruptions and gene replacements to mutagenically characterize 30 kilobases of DNA in the erythromycin resistance gene (ermE) region of the Saccharopolyspora erythraea chromosome. Five previously undiscovered loci involved in the biosynthesis of erythromycin were found, eryBI, eryBII, eryCI, eryCII, and eryH; and three known loci, eryAI, eryG, and ermE, were further characterized. The new Ery phenotype, EryH, was marked by (i) the accumulation of the intermediate 6-deoxyerythronolide B (DEB), suggesting a defect in the operation of the C-6 hydroxylase system, and (ii) a block in the synthesis or addition reactions for the first sugar group. Analyses of ermE mutants indicated that ermE is the only gene required for resistance to erythromycin, and that it is not required for production of the intermediate erythronolide B (EB) or for conversion of the intermediate 3-alpha-mycarosyl erythronolide B (MEB) to erythromycin. Mutations in the eryB and eryC loci were similar to previously reported chemically induced eryB and eryC mutations blocking synthesis or attachment of the two erythromycin sugar groups. Insertion mutations in eryAI, the macrolactone synthetase, defined the largest (at least 9-kilobase) transcription unit of the cluster. These mutants help to define the physical organization of the erythromycin gene cluster, and the eryH mutants provide a source for the production of the intermediate DEB.

Mutation and cloning of eryG, the structural gene for erythromycin O-methyltransferase from Saccharopolyspora erythraea, and expression of eryG in Escherichia coli.

A mutant strain derived by chemical mutagenesis of Saccharopolyspora erythraea (formerly known as Streptomyces erythreus) was isolated that accumulated erythromycin C and, to a lesser extent, its precursor, erythromycin D, with little or no production of erythromycin A or erythromycin B (the 3"-O-methylation products of erythromycin C and D, respectively). This mutant lacked detectable erythromycin O-methyltransferase activity with erythromycin C, erythromycin D, or the analogs 2-norerythromycin C and 2-norerythromycin D as substrates. A 4.5-kilobase DNA fragment from S. erythraea originating approximately 5 kilobases from the erythromycin resistance gene ermE was identified that regenerated the parental phenotype and restored erythromycin O-methyltransferase activity when transformed into the erythromycin O-methyltransferase-negative mutant. Erythromycin O-methyltransferase activity was detected when the 4.5-kilobase fragment was fused to the lacZ promoter and introduced into Escherichia coli. The activity was dependent on the orientation of the DNA relative to lacZ. We have designated this genotype eryG in agreement with Weber et al. (J.M. Weber, B. Schoner, and R. Losick, Gene 75:235-241, 1989). It thus appears that a single enzyme catalyzes all of the 3"-O-methylation reactions of the erythromycin biosynthetic pathway in S. erythraea and that eryG codes for the structural gene of this enzyme.

Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay.

We have evaluated the diagnostic utility of eleven Toxoplasma gondii recombinant antigens (P22 [SAG2], P24 [GRA1], P25, P28 [GRA2], P29 [GRA7], P30 [SAG1], P35, P41 [GRA4], P54 [ROP2], P66 [ROP1], and P68) in immunoglobulin G (IgG) and IgM recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Following an initial evaluation, six recombinant antigens (P29, P30, P35, P54, P66, and P68) were tested in the IgG and IgM Rec-ELISAs with four groups of samples which span the toxoplasmosis disease spectrum (negative, chronic infection, acute infection, and recent seroconversion). Our results suggest that the combination of P29, P30, and P35 in an IgG Rec-ELISA and the combination of P29, P35, and P66 in an IgM Rec-ELISA can replace the tachyzoite antigen in IgG and IgM serologic tests, respectively. The relative sensitivity, specificity, and agreement for the IgG P29-P30-P35 Rec-ELISA were 98.4, 95.7, and 97.2%, respectively. The resolved sensitivity, specificity, and agreement for the IgM P29-P35-P66 Rec-ELISA were 93.1, 95.0, and 94. 5%, respectively. Relative to the tachyzoite-based immunocapture IgM assay, the IgM P29-P35-P66 Rec-ELISA detects fewer samples that contain IgG antibodies with elevated avidity from individuals with an acute toxoplasmosis.

Development of a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot for detection of CMV-specific IgM.

We developed a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot to detect CMV-specific IgM in human sera. The new test contains four viral proteins (vp150, vp82, vp65, and vp28) purified from viral particles and four recombinant proteins (rp150, rp130, rp52, and rp38) purified from Escherichia coli. These antigens were individually loaded onto nitrocellulose strips, and the strips were then used to detect CMV-specific IgM by using a mu-specific conjugate. The new assay was evaluated in parallel with one or two IgM enzyme-linked immunosorbent assays (ELISAs) to test 592 serum samples from different groups of latently or acutely infected individuals. The sensitivity of the new assay with respect to the consensus of two ELISAs was 100%, the specificity was 98.6%, the positive predictive value was 96.9%, and the negative predictive value was 100%. We also evaluated the new test by testing sera from pregnant women and transplant recipients with a known clinical history. Our results suggest that the new test combines high sensitivity with high specificity, characteristics that are mutually exclusive with the other commercially available tests. Furthermore, a statistically significant correlation was observed between the number of IgM-reactive bands and the elevated risk of transmission from CMV-infected pregnant women to their offspring.

Evaluation of the Abbott AxSYM cytomegalovirus (CMV) immunoglobulin M (IgM) assay in conjunction with other CMV IgM tests and a CMV IgG avidity assay.

The measurement of the avidity of cytomegalovirus (CMV) immunoglobulin G (IgG) antibodies has been shown by several investigators to be useful in identifying and excluding primary CMV infections in pregnant women. In this work, we examined the diagnostic utility of reflex testing of CMV IgM-positive specimens from pregnant women by using a CMV IgG avidity assay. The utility of this approach was directly dependent on the sensitivity of the CMV IgM assay employed during the initial screen. The higher initial reactivity rate of the AxSYM CMV IgM assay was necessary in order to detect CMV IgM in specimens containing low-avidity CMV IgG antibodies, indicative of a primary CMV infection, which other CMV IgM assays (Behring, Vidas, Captia, and Eurogenetics) fail to detect in some cases. The use of the AxSYM CMV IgM assay, followed by an avidity test, should result in more accurate diagnosis of CMV infection in pregnant women.

Relationship between IgM antibody to human cytomegalovirus, virus load, donor and recipient serostatus, and administration of methylprednisolone as risk factors for cytomegalovirus disease after liver transplantation.

A retrospective study was performed on a selected cohort of 40 liver transplant recipients derived from the previous prospective follow-up of 162 liver transplant patients. The criterion for selection of this cohort was the presence of human cytomegalovirus (HCMV) DNAemia after transplantation, as determined by qualitative polymerase chain reaction (PCR). These 40 patients were followed longitudinally by quantitative PCR and by the new recombinant antigen-based AxSYM immunoassay for IgM to HCMV. The detection of IgM to CMV after transplantation was significantly associated with the development of HCMV disease in patients who had evidence of active HCMV replication in the blood by PCR (P=.01). On the basis of multivariate logistic regression analyses, the maximum titer of IgM detected after transplantation was a risk factor that was independent of augmented methylprednisolone and donor seropositivity. However, in multivariate analyses, elevated virus load continued to be the predominant risk factor for progression to HCMV disease.

Development and clinical evaluation of a recombinant-antigen-based cytomegalovirus immunoglobulin M automated immunoassay using the Abbott AxSYM analyzer.

A new microparticle enzyme immunoassay (MEIA), the Cytomegalovirus (CMV) Immunoglobulin M (IgM) test, was developed on the Abbott AxSYM analyzer. This test uses recombinant CMV antigens derived from portions of four structural and nonstructural proteins of CMV: pUL32 (pp150), pUL44 (pp52), pUL83 (pp65), and pUL80a (pp38). A total of 1, 608 specimens from random volunteer blood donors (n = 300), pregnant women (n = 1,118), transplant recipients (n = 6), and patients with various clinical conditions and disease states (n = 184) were tested during development and evaluation of this new assay. In a preliminary clinical evaluation we tested specimens collected prospectively from pregnant women (n = 799) and selected CMV IgM-positive archived specimens from pregnant women (n = 39). The results from the new CMV IgM immunoassay were compared to the results of a consensus interpretation of the results obtained with three commercial CMV IgM immunoassays. The results for specimens with discordant results were resolved by a CMV IgM immunoblot assay. The relative sensitivity, specificity, and agreement for the AxSYM CMV IgM assay were 94.29, 96.28, and 96.19%, respectively, and the resolved sensitivity, specificity, and agreement were 95.83, 97.47, and 97.37%, respectively. We also tested serial specimens from women who experienced seroconversion or a recent CMV infection during gestation (n = 17) and potentially cross-reactive specimens negative for CMV IgM antibody by the consensus tests (n = 184). The AxSYM CMV IgM assay was very sensitive for the detection of CMV IgM during primary CMV infection, as shown by the detection of CMV IgM at the same time as or just prior to the detection of CMV IgG. Specimens from individuals with lupus (n = 16) or parvovirus B19 infection (n = 6) or specimens containing hyper IgM (n = 9), hyper IgG (n = 8), or rheumatoid factor (n = 55) did not cross-react with the AxSYM assay. One specimen each from individuals infected with Epstein-Barr virus (n = 26), measles virus (n = 10), herpes simplex virus (n = 12), or varicella-zoster virus (n = 13) infection, one specimen from an influenza vaccinee (n = 14), and one specimen containing antinuclear antibody cross-reacted with the assay. The overall rate of cross-reactivity of the specimens with the assay was 3.3% (6 of 184). The AxSYM CMV IgM assay is a sensitive and specific assay for the detection of CMV-specific IgM.