Purinergic receptors have been found to modulate ion transport in several types of epithelial cells as well as excitable cells. It was of interest to determine whether vestibular dark cells and strial marginal cells contain purinergic receptors in either the apicalor basolateral membrane which modulate transepithelial ion transport. Vestibular dark cell and strial marginal cell epithelia were mounted in a micro-Ussing chamber for the measurement of the transepithelial voltage and resistance from which the equivalent short circuit current (Isc) was obtained. The apical and basolateral sides were independently perfused with adenosine and adenosine 5′-triphosphate (ATP). Adenosine (10−5 M) had no effect on Isc at either the apical or basolateral side of vestibular dark cells and strial marginal cells, suggesting either the absence of P1 receptors or the absence of coupling of P1 receptors to vectorial ion transport by these epithelia. Apical perfusion of ATP (10−8 to 10−4 M) caused a decrease in Isc of both vestibular dark cells and strial marginal cells. Apical perfusion of the nucleotides uridine 5′-triphosphate (UTP), 2-methylthioadenosine triphosphate (2-meS-ATP), adenosine 5′-O-(3-thiotriphosphate) (ATPγS) and α,β-methylene adenosine 5′-triphosphate (α,β-meth-ATP) caused qualitatively similar responses with different magnitudes of response. The sequence of the magnitude of response of each compound at 10−6 or 10−5 M was assessed from the fractional change of Isc. The sequence for vestibular dark cells was UTP = ATP = ATPγS ≫ 2-meS-ATP > α,β-meth-ATP, and for strial marginal cells it was UTP = ATP ≫ 2-meS-ATP, corresponding to the sequence for the P2U receptor. The effect of agonist on the apical membrane was reduced by the antagonist 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) but not cibacron blue or suramin. DIDS in the absence of exogenous purinergic agonist caused a sustained increase in Isc. The effect of ATP on the apical membrane was greater in the absence of divalent cations. Basolateral perfusion of ATP led to a biphasic response of Isc in vestibular dark cell and strial marginal cell epithelia, consisting of an initial rapid increase followed by a slower decrease. Perfusion of the perilymphatic surface of the stria vascularis (basal cell layer) with ATP had no acute effect on Isc. The initial increase of Isc in vestibular dark cell epithelium during basolateral perfusion had a sequence of 2-meS-ATP > ATP ≫ UTP = α,β-meth-ATP = ATPγS, corresponding to the sequence for the P2Y receptor. Subsequently, the agonists caused a sustained decrease in Isc with a sequence of ATPγS > 2-meS-ATP > ATP > UTP >α,β-meth-ATP. This sequence is most simply interpreted as the result of the coexistence of P2U and P2Y receptors in the basolateral membrane. Both the increase and decrease of Isc by ATP at the basolateral membrane were reduced by the antagonist suramin. These findings provide evidence for the regulation of transepithelial ion transport by P2U receptors in the apical membrane and by coexisting P2U and P2Y receptors in the basolateral membrane of K+-secretory epithelial cells in the inner ear and are consistent with the hypothesis that the apical receptors are part of an autocrine negative feedback system in these cells.
P2U receptor; P2 agonists; adenosine; DIDS; cibacron blue; suramin; reactive blue 2
A non-trivial cluster growth model, equivalent to the lattice-free Eden-C model, is proposed. The model is constructed by randomly adding contiguous circles without overlapping. Large-scale computer simulations show the interior density is constant at 0.650, while the boundary is fractal, with a thickness proportional to the 0.396 power of the mean radius.
Race, ethnicity, and cultural attitudes and practices are among the variables that influence health behaviors, including adaptive health behaviors. The following discussions highlight the important role of social conditions in shaping health behaviors and the central role of family in promoting health across the Asian, Hispanic, Native American, and African American ethnic groups. Factors that may lead to health-damaging behaviors are also discussed. The need for additional research that identifies correlations among physiological, social, and behavioral factors and health behaviors, as well as underlying mechanisms, is called for.
aging; behavior; coping; ethnicity; health
OBJECTIVE--To describe the clinical expression of primary Sjögren's syndrome (SS) in men, focusing on extraglandular manifestations (EGM) and serological markers of disease. METHODS--In a cross sectional and comparative study, adult men with primary SS were identified from a cohort study on SS, and 26 age matched adult women with primary SS were selected as a control group. All patients met the European classification criteria for SS. They were compared for demographic, clinical and laboratory findings. RESULTS--Thirteen men with primary SS were identified. Mean age at onset was 39 (SEM 4) years and mean duration of disease was 7.8 (1) years. Sicca complex or parotitis was the presenting feature in eight patients (61.5%), and an EGM in five (38.5%). During the course of the disease, EGM were present in 12 patients (92%), polyarthralgias and lymphopenia being the most frequent (38.5% each). Rheumatoid factor was positive in 73% of patients, antinuclear antibodies in 85%, anti-(SS-A) in 62%, and anti-(SS-B) in 46%. No statistical differences in the frequency of EGM or in the presence of autoantibodies were observed between men and women. However, men patients were more likely to have EGM. CONCLUSION--Primary SS in men is an uncommon condition with clinical and serological characteristics similar to those observed in women. Sex hormones may be incriminated in the pathogenesis of SS. However, it remains poorly understood whether sex hormones play a major role in the severity of disease and have any importance with regard to treatment.
A 3-year study of spotted fever group rickettsial ecology in Inner Mongolia revealed that nearly half of the human population tested had antibodies to Rickettsia sibirica detected by complement fixation test. Infected persons, ticks and a high proportion of seropositive livestock and wild rodents were found in all five vegetation zones (desert, steppe, forest, forest-grassland and grassland).
Specific killing of erbB-2-overexpressing tumor cells can be achieved using expression of an intracellular antibody directed against the erbB-2 oncoprotein. We have developed a strategy using a recombinant adenovirus encoding an anti-erbB-2 single chain antibody to achieve targeted tumor cell killing in vivo and can show significantly prolonged survival of animals carrying a human ovarian carcinoma tumor burden within their peritoneal cavities. This strategy of gene therapy for ovarian carcinoma offers the potential to achieve highly specific, targeted killing of human tumor cells and thus establishes the rationale to undertake human clinical trials on this basis.
Intimal thickening after vascular injury may be modulated in part by heparin binding growth factors. We hypothesized that placement of a therapeutic polymer in the periadventitial space capable of tightly binding growth factors might alter the vascular response to injury. We first demonstrated that incubation of rat aortic smooth muscle cells with an insoluble, sulfated polymer of beta-cyclodextrin (P-CDS) was associated with a dose-dependent inhibition of proliferation induced by fetal calf serum, fibroblast growth factor-2 (FGF-2), platelet-derived growth factor BB, or epidermal growth factor. Preincubation studies of P-CDS with FGF-2 revealed a very rapid removal of mitogenic activity. Using radiolabeled FGF-2 (0.25 microg/ml), we observed a very rapid association rate (0.34 +/- 0.07 min-1, n=4) and a very slow dissociation rate (3.3 +/- 0.2 X 10(-7) min-1) at 37 degrees C, suggesting a high affinity interaction. Using both Transwell and linear under-agarose assays, we demonstrated a significant inhibition of random migration (chemokinesis) by P-CDS. Unsulfated polymeric beta-cyclodextrin (P-CD) had little if any of these effects, suggesting that the high negative charge density of P-CDS was important for the effects. Finally, rats undergoing carotid artery balloon injury were randomized to treatment with periadventitial P-CDS or no treatment, and were killed at 4 (n=20), 14 (n=59), and 88 d (n=14). Morphometric analysis demonstrated significant and sustained inhibition of intimal thickening in P-CDS-treated rats at 14 (P < 0.01) and 88 d (P < 0.05) using absolute intimal area or intima/media area ratios. No inhibition was seen in a group of rats treated with P-CD. In P-CDS-treated rats, bromodeoxyuridine labeling studies revealed fewer labeled smooth muscle cells in the intima at 14 d (P=0.01), while staining with Evans blue revealed enhanced late endothelial cell regrowth. Thus, periadventitially applied sulfated beta-cyclodextrin polymer, which can tightly bind heparin binding growth factors, inhibits intimal thickening in vivo in a sustained fashion without using an additional delivery system. These studies suggest that cellular processes mediated by heparin binding growth factors may be modulated by P-CDS.
In this study, we evaluated three PCR methods for epidemiological typing of Burkholderia (Pseudomonas) cepacia--PCR-ribotyping, arbitrarily primed PCR (AP-PCR) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR)--and compared them with pulsed-field gel electrophoresis. The analysis was performed with 31 isolates of B. cepacia, comprising 23 epidemiologically unrelated isolates and 8 isolates collected from the same patient during two episodes of bacteremia. Pulsed-field gel electrophoresis, ERIC-PCR, and AP-PCR identified 23 distinct types among the 23 unrelated isolates, while PCR-ribotyping only identified 12 strain types, even after AluI digestion of the amplification products. Among the eight isolates collected from the same patient, all typing techniques revealed two clones of strains. The day-to-day reproducibilities of PCR-ribotyping and ERIC-PCR were good, while greater day-to-day variations were noted in the fingerprints obtained by AP-PCR. We conclude that all three PCR techniques are useful for rapid epidemiological typing of B. cepacia, but ERIC-PCR seems to be more reproducible and discriminative.
We determined whether the infectivity of the Lyme disease spirochete (Borrelia burgdorferi) to vector ticks varies with the duration of infection in laboratory mice. Thus, noninfected nymphal deer ticks were permitted to feed on two strains of early (2 months after infection) and late (8 months after infection) spirochete-infected mice. The attached ticks were removed from their hosts at specified time intervals and were thereafter examined for spirochetes by direct immunofluorescence microscopy. Spirochetes can be acquired by nymphal ticks as fast as 8 h after attachment. More than 80% of the attached ticks acquired spirochetal infection within 48 h after feeding on early spirochete-infected mice. In contrast, spirochetal infectivity to ticks was less than 50% after feeding on late spirochete-infected mice. The overall infectivity of spirochete-infected mice to ticks correlated with the duration of tick attachment. In addition, there was no adverse effect on the spirochetal infectivity to ticks by high levels of host antibody against spirochetes, and no obvious differences in infectivity to ticks was observed by the site of tick feeding. We conclude that the span of spirochetal infectivity to ticks varies with the duration of infection in mice and suggest that spirochetes may persist and may be evenly distributed in the skin of infected hosts, regardless of prominent host immunity.
OBJECTIVES--A cross sectional study was performed to find the concentrations of elements contained in the semen of workers exposed to trinitrotoluene (TNT). SUBJECTS AND METHODS--Semen of exposed workers in two TNT plants located in He-Nan Province in 1992 were examined. RESULTS--The average TNT concentrations in the workplace, except the packing site, were found to have exceeded the maximal allowable concentration (MAC, 1 mg/m3); skin contaminations of male workers exposed to TNT were higher after a shift than in controls, and correlated with the total blood concentrations of TNT, 4-amino-2, 6-dinitrotoluene (4A), and 2-amino-4, 6-dinitrotoluene (2A). Cu, Zn, Na, Mg, and Se concentrations were significantly decreased, but K, Ca, Co, Mn and Li contents were not significantly changed in the semen of workers exposed to TNT. Compared with the control group, the percentage of liquifying time of semen, the sperm malformation incidence, and viability in the men exposed to TNT were all significantly changed. CONCLUSIONS--Men exposed to TNT have decreased concentrations of some elements is semen and altered semen physiology.
The vif gene of human and simian immunodeficiency viruses (HIV and SIV) encodes a late gene product that is essential for viral infectivity in natural target cells. Virions produced in the absence of Vif are abnormal in their ultrastructural morphology and are severely impaired in the ability to complete proviral DNA synthesis upon entry into new target cells. Because previous studies failed to detect Vif protein in virus particles, Vif is believed to influence virus infectivity indirectly, by affecting virion assembly, release, and/or maturation. In this report, we reexamined the possibility that Vif is a virion-associated protein. Utilizing high-titer Vif-specific antibodies, a sensitive immunoblot technique, and highly concentrated virus preparations, we detected a 23-kDa Vif-reactive protein in wild-type HIV type 1 (HIV-1) and a 27-kDa Vif-reactive protein in wild-type SIVSM virions. Neither protein was present in virions derived from vif-deficient HIV-1 and SIVSM proviral constructs. Vif protein content was similar among different strains of HIV-1 and was independent of the cell type (permissive or nonpermissive) used to produce the virus. To determine the subvirion localization of Vif, HIV-1 virions were treated with proteinase K or Triton X-100 to remove virion surface proteins and the viral membrane, respectively, purified through sucrose, and analyzed by immunoblot analysis. Vif protein content was not affected by the removal of external surface proteins or by the removal of the viral membrane and submembrane p17Gag matrix protein. Instead, Vif colocalized with viral core structures which sedimented at a density of 1.25 g/ml on linear sucrose gradients (enveloped HIV-1 particles sediment at a density of 1.17 g/ml). Finally, the amount of Vif protein packaged into virions was estimated to be on the order of 1 molecule of Vif for every 20 to 30 molecules of p24Gag, or between 60 and 100 molecules of Vif per particle. These results indicate that Vif represents an integral component of HIV and SIV particles and raise the possibility that it plays a direct role in early replication events.
Erythromycin is the drug of choice for treatment of Mycoplasma pneumoniae infections due to its susceptibility to low levels of this antibiotic. After exposure of susceptible strains to erythromycin in vitro and in vivo, mutants resistant to erythromycin and other macrolides were isolated. Their phenotypes have been characterized, but the genetic basis for resistance has never been determined. We isolated two resistant mutants (M129-ER1 and M129-ER2) by growing M. pneumoniae M129 on agar containing different amounts of erythromycin. In broth dilution tests both strains displayed resistance to high levels of several macrolide-lincosamide-streptogramin B (MLS) antibiotics. In binding studies, ribosomes isolated from the resistant strains exhibited significantly lower affinity for [14C]erythromycin than did ribosomes from the M129 parent strain. Sequencing of DNA amplified from the region of the 2S rRNA gene encoding domain V revealed an A-to-G transition in the central loop at position 2063 of M129-ER1 and a similar A-to-G transition at position 2064 in M129-ER2. Transitions at homologous locations in the 23S rRNA from other organisms have been shown to result in resistance to MLS antibiotics. Thus, MLS-like resistance can occur in M. pneumoniae as the result of point mutations in the 23S rRNA gene which reduce the affinity of these antibiotics for the ribosome. Since they involve only single-base changes, development of resistance to erythromycin in vivo by these mechanisms could be relatively frequent event.
Dihydrouracil (DHU) is a major base damage product formed from cytosine following exposure of DNA to ionizing radiation under anoxic conditions. To gain insight into the DNA lesion structural requirements for RNA polymerase arrest or bypass at various DNA damages located on the transcribed strand during elongation, DHU was placed onto promoter-containing DNA templates 20 nucleotides downstream from the transcription start site. In vitro, single-round transcription experiments carried out with SP6 and T7 RNA polymerases revealed that following a brief pause at the DHU site, both enzymes efficiently bypass this lesion with subsequent rapid generation of full-length runoff transcripts. Direct sequence analysis of these transcripts indicated that both RNA polymerases insert primarily adenine opposite to the DHU site, resulting in a G-to-A transition mutation in the lesion bypass product. Such bypass and insertion events at DHU sites (or other types of DNA damages), if they occur in vivo, have a number of important implications for both the repair of such lesions and the DNA damage-induced production of mutant proteins at the level of transcription (transcriptional mutagenesis).
The genetic complexity of vaccinia virus is such that as well as encoding its own transcription and replication machinery, it encodes two protein kinases and a protein phosphatase. The latter enzyme, designated VH1, is a prototype for the dual-specificity class of phosphatases. Here we report that the H1 phosphatase is encapsidated within vaccinia virions and describe the construction of a viral recombinant in which expression of the H1 gene is regulated by the presence or absence of isopropylthiogalactopyranoside (IPTG) in the culture medium. When expression of H1 is repressed, the number of viral particles produced is not compromised but the fraction of these particles which is infectious is significantly reduced. The lack of infectivity of the H1-deficient particles is specifically correlated with their inability to direct the transcription of early genes either in vitro or in vivo. A proximal role for the viral phosphatase in regulating the onset of viral gene expression is implied. Prominent among the encapsidated proteins found to be hyperphosphorylated in H1-deficient virions is the 11-kDa product of the F18 gene; this protein is the major DNA-binding component of the viral nucleoprotein complex. The ability of recombinant H1 phosphatase to reverse this hyperphosphorylation in permeabilized virions strengthens the conclusion that the F18 protein is a bona fide substrate for the H1 phosphatase.
In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05). Immunohistochemical analysis as well as in situ hybridization showed that inducible NO synthase was predominantly expressed in synovial lining cells, endothelial cells, chondrocytes, and to a lesser extent, in infiltrating mononuclear cells and synovial fibroblasts. The synovial lining cells and the infiltrating cells expressing inducible NO synthase were identified where CD14+ cells were located. Together with morphological features, this suggests that they are type A synoviocytes. NO production from freshly isolated synoviocytes and chondrocytes was up-regulated by in vitro stimulation with a combination of IL-TNF-beta, TNF-alpha, and LPS. In summary, the present results suggest that NO is produced primarily by CD14+ synoviocytes, chondrocytes, and endothelial cells in inflammatory joints of arthritides. NO production can be upregulated by cytokines present in inflamed joints. The increased NO production may thus contribute to the pathological features in inflammatory arthritides.
The dnrQS genes from the daunorubicin producer Streptomyces peucetius were characterized by DNA sequencing, complementation analysis, and gene disruption. The dnrQ gene is required for daunosamine biosynthesis, and dnrS appears to encode a glycosyltransferase for the addition of the 2,3,6-trideoxy-3-aminohexose, daunosamine, to epsilon-rhodomycinone.
The N(inf2)-fixing bacterium Azotobacter vinelandii was grown in an O(inf2)-regulated chemostat with glucose or galactose as substrate. Increasing the O(inf2) partial pressure resulted in identical synthesis of the noncoupled cytochrome d terminal oxidase, which is consistent with the hypothesis that A. vinelandii uses high rates of respiration to protect the nitrogenase from oxygen. However, cell growth on glucose showed a lower yield of biomass, higher glycolytic rate, higher respiratory rate, and lower cytochrome o content than cell growth on galactose. Elemental analysis indicated no appreciable change in the C-to-N ratio of cell cultures, suggesting that the major composition of the cell was not influenced by the carbon source. A poor coordination of glucose and nitrogen metabolisms in A. vinelandii was suggested. The rapid hydrolysis of glucose resulted in carbonaceous accumulation in cells. Thus, Azotobacter species must induce a futile electron transport to protect cells from the high rates of glucose uptake and glycolysis.
Salmonella paratyphi A, a human-adapted bacterial pathogen, causes paratyphoid enteric fever. We established the genome map of strain ATCC 9150 by the use of four endonucleases, XbaI, I-CeuI, AvrII (= BlnI), and SpeI, which generated 27, 7, 19, and 38 fragments, respectively; the sum of the fragments in each case indicates a genome size of ca. 4,600 kb. With phage P22, we transduced Tn10 insertions in known genes from Salmonella typhimurium LT2 to S. paratyphi A ATCC 9150 and located these insertions on the S. paratyphi A chromosome through the XbaI and AvrII sites in Tn10 and through the increased size of the SpeI fragment bearing a Tn10. Compared with the maps of other Salmonella species, the S. paratyphi A genomic map showed two major differences: (i) an insertion of about 100 kb of DNA between rrnH/G and proB and (ii) an inversion of half the genome between rrnH and rrnG, postulated to be due to homologous recombination between the rrn genes. We propose that during the evolution of S. paratyphi A, the first rearrangement event was the 100-kb insertion, which disrupted the chromosomal balance between oriC and the termination of replication, forcing the rrnH/G inversion to restore the balance. The insertion and the inversion are both present in all 10 independent wild-type S. paratyphi A strains tested.
p53, the protein encoded by one of the most significant human tumor suppressor genes, is a sequence-specific transcriptional activator. When activated by a double-stranded DNA break, p53 function arrests cells in G1 and can induce apoptosis. Transcriptional activation function is critical for p53 tumor suppression, although transcriptional repressing and nontranscriptional functions of p53 may contribute. p53 activation requires that it bind to TFIID through interactions with TATA box-binding protein (TBP)-associated factors and potentially with TBP. Here, we studied the mechanism of p53 activation using in vitro transcription and a sufficiently high p53 concentration to squelch activated transcription. Squelching is thought to result when target molecules that interact with activation domains are titrated by binding to excess activator. Addition of either excess TFIIB or TFIID but not other proteins required for p53-activated transcription reversed squelching by high p53 concentrations, whereas neither stimulated transcription in reactions without excess p53. These results reveal that both TFIIB and TFIID are inhibited by high concentrations of p53 and suggest that p53 activation may work through direct or indirect interactions with both TFIIB and TFIID.
In a 6-microns capillary filled with buffer and in the absence of any chemotactic stimuli, Escherichia coli K-12 cells swim persistently in only one direction. This behavior of E. coli can be simply explained by means of the length and relative rigidity of their flagella. Single-cell motility parameters--swimming speed, turn angle, and run length time--were measured. Compared with the motility parameters measured in bulk phase, turn angle was influenced because of the effect of the geometrical restriction.
The gene products from an 8-kb region adjacent to the 3' end of the ptx operon are required by Bordetella pertussis for the export of pertussis holotoxin. At least one of these gene products (PtlC) is specifically required for the export of assembled holotoxin from the periplasmic space. ptlC mutants exhibit a 20-fold reduction in the amount of holotoxin present in the culture supernatant but have no effect upon the assembly or steady-state level of holotoxin present in the periplasmic space. Impaired export of holotoxin from the ptlC strain blocks expression of toxin at a posttranscriptional level, and wild-type levels of ptx mRNA are detected in the mutant strain. The transcription of ptl is subject to modulation by MgSO4 in the same manner as ptx is; however, in B. pertussis strains containing an E. coli tac promoter in place of the native ptx promoter, wild-type levels of ptx mRNA are present and holotoxin is synthesized and exported even in the presence of MgSO4. Promoter mapping of the region extending from the ptxS3 coding region to the ptlC coding region failed to detect the ptl transcription initiation site. Additional RNase protection experiments with ptx promoter deletion and substitution strains indicate that the ptl operon is transcribed from the ptx promoter as part of a > 11-kb mRNA.
Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.
Vaccinia virus encodes two protein kinases; the B1 kinase is expressed early and appears to play a role during DNA replication, whereas the F10 kinase is expressed late and is encapsidated in virions. Here we report that the F10 kinase gene is the locus affected in a complementation group of temperature-sensitive mutants composed of ts15, ts28, ts54, and ts61. Although these mutants have a biochemically normal phenotype at the nonpermissive temperature, directing the full program of viral gene expression, they fail to form mature virions. Electron microscopic analysis indicates that morphogenesis undergoes arrest at a very early stage, prior to the formation of membrane crescents or immature virions. An essential role for the F10 protein kinase in orchestrating the onset of virion assembly is implied.
Ubiquitin-conjugating (E2) enzymes contain several regions within their catalytic domains that are highly conserved. However, within some of these conserved regions are several residues that may be used to define different classes of catalytic domains for the E2 enzymes. One class can be defined by the Ubc1 protein, which contains K-65, D-90, and D-120, while the corresponding positions within the Cdc34 (Ubc3) protein, which defines a second class of enzymes, contain S-73, S-97, and S-139, respectively. The presence of these differences within otherwise highly conserved regions of this family suggests that these residues may be critical for the specificity of Cdc34 function or regulation. Therefore, we have constructed a series of cdc34 alleles encoding mutant proteins in which these serine residues have been changed to other amino acid residues, including alanine and aspartic acid. In vivo complementation studies showed that S-97, which lies near the active site C-95, is essential for Cdc34 function. The addition of a second mutation in CDC34, which now encoded both the S97D and S73K changes, restored partial function to the Cdc34 enzyme. Moreover, the deletion of residues 103 to 114 within Cdc34, which are not present in the Ubc1-like E2s, allowed the S73K/S97D mutant to function as efficiently as wild-type Cdc34 protein. Finally, the cloning and sequencing of the temperature-sensitive alleles of CDC34 indicated that A-62 is also unique to the Cdc34 class of E2 enzymes and that mutations at this position can be detrimental to Cdc34 function. Our results suggest that several key residues within conserved regions of the E2 enzyme family genetically interact with each other and define a class of E2 catalytic domains.