Fine Mapping of the Gene for Carbohydrate-Deficient Glycoprotein Syndrome, Type I (CDG1): Linkage Disequilibrium and Founder Effect in Scandinavian Families

Carbohydrate-deficient glycoprotein syndrome type I (CDG I) is characterized clinically by severe nervous system involvement and biochemically by defects in the carbohydrate residues in a number of serum glycoproteins. The CDG1 gene was recently localized by us to a 13-cM interval in chromosome region 16p13. In this study 44 CDG I families from nine countries were analyzed with available markers in a region ranging from marker D16S495 to D16S497, and haplotype and linkage disequilibrium analyses were performed. One specific haplotype was found to be markedly overrepresented in CDG I patients from a geographically distinct region in Scandinavia, strongly indicating that CDG I families in this region share the same ancestral CDG1 mutation. Furthermore, analysis of the extent of the common haplotype in these families indicates that the CDG1 gene is located in the region defined by markers D16S513–AFMa284wd5–D16S768–D16S406–D16S502. The critical CDG1 region, in strong linkage disequilibrium with markers AFMa284wd5, D16S768, and D16S406, thus constitutes less than 1 Mb of DNA and less than 1 cM in the very distal part of the CDG1 region defined by us previously.     

Oral Ingestion of Mannose Elevates Blood Mannose Levels: A First Step toward a Potential Therapy for Carbohydrate-Deficient Glycoprotein Syndrome Type I

Carbohydrate-deficient glycoprotein syndrome type I (CDGS) is an inherited metabolic disorder with multisystemic abnormalities resulting from a failure to add entire N-linked oligosaccharide chains to many glycoproteins. Fibroblasts from these patients also abnormally glycosylate proteins, but this lesion is corrected by providing 250 μm mannose to the culture medium. This correction of protein glycosylation suggests that providing dietary mannose to elevate blood mannose concentrations might also remedy some of the underglycosylation observed in these patients. We find that ingested mannose is efficiently absorbed and increases blood mannose levels in both normal subjects and CDGS patients. Blood mannose levels increased in a dose-dependent fashion with increasing oral doses of mannose (0.07–0.21 g mannose/kg body weight). Peak blood mannose concentrations occurred at 1–2 h following ingestion and the clearance half-time was approximately 4 h. Doses of 0.1 g mannose/kg body weight given at 3-h intervals maintained blood mannose concentrations at levels 3- to 5-fold higher than the basal level in both normal controls (∼55 μm) and CDGS patients. No side effects were observed for this dosage regimen. These results establish the feasibility of using mannose as a potential therapeutic dietary supplement (nutraceutical) to treat CDGS patients.     

Evidence for an Adenovirus Type 2-Coded Early Glycoprotein

We have identified an adenovirus type 2 (Ad2)-induced early glycopolypeptide with an apparent molecular weight of 20,000 to 21,000 (20/21K), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 20/21K polypeptide could be labeled in vivo with [(3)H]glucosamine. [(35)S]methionine- and [(3)H]-glucosamine-labeled 20/21K polypeptides bound to concanavalin A-Sepharose columns and were eluted with 0.2 M methyl-alpha-d-mannoside. The pulse-labeled polypeptide appeared as a sharp band with an apparent molecular weight of 21K, but after a chase it converted to multiple bands with an average molecular weight of 20K. This variability in electrophoretic mobility is consistent with glycosylation or deglycosylation of the 20/21K polypeptide. Analysis of the pulse and pulse-chase-labeled forms by using partial proteolysis indicated that the polypeptides were highly related chemically, but not identical. Most of the 20/21K polypeptide is localized in the cytoplasm fraction of infected cells lysed by Nonidet P-40. The 20/21K polypeptide and a 44K polypeptide, labeled with [(35)S]methionine or [(3)H]glucosamine in Ad2-infected human cells, were precipitated by a rat antiserum against an Ad2-transformed rat cell line (T2C4), but not by antisera against three other Ad2-transformed rat cell lines, or by serum from nonimmune rats. The partial proteolysis patterns of the 20/21K and the 44K polypeptides were indistinguishable, indicating that the two polypeptides are highly related, and suggesting that the 44K polypeptide might be a dimer of the 20/21K polypeptide. The 20/21K polypeptide was also induced in Ad2-early infected monkey and hamster cells. These results imply that the 20/21K polypeptide is synthesized in Ad2-infected human, monkey, and hamster cells, and in one but not all Ad2-transformed rat cells. Thus, the 20/21K polypeptide is probably viral coded rather than cell coded and viral induced.     

Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9

Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.Alphaherpesviruses are parasites of the peripheral nervous system. In their natural hosts, alphaherpesviruses establish lifelong persistent infections in sensory ganglia and periodically return by axonal transport to the periphery, where they cause recurrent disease. This life cycle requires viral transport along axons in two directions. Axonal transport in the retrograde direction (toward the neuron cell body) occurs during neuroinvasion and is required for the establishment of latency, while transport in the anterograde direction (away from the neuron cell body) occurs after reactivation and is required for viral spread to the periphery to cause recurrent disease. In addition to anterograde and retrograde axonal transport within neurons, alphaherpesviruses spread between synaptically connected neurons and between neurons and epithelial cells at the periphery (19, 22).The alphaherpesvirus subfamily includes the human pathogens herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), as well as numerous veterinary pathogens such as pseudorabies virus (PRV) and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5). The molecular mechanisms that mediate alphaherpesvirus anterograde axonal transport, anterograde spread, and cell-to-cell spread remain unclear. However, many studies of several alphaherpesviruses have indicated that anterograde transport or anterograde spread involves the viral proteins glycoprotein E (gE), glycoprotein I (gI), and Us9 (2, 5, 7, 9, 11, 13, 16, 26, 30, 31, 41, 46, 51, 52).Glycoproteins E and I are type I membrane proteins that form a heterodimer in the virion membrane and on the surface of infected cells. Although dispensable for the entry of extracellular virus, gE and gI mediate the epithelial cell-to-cell spread of numerous alphaherpesviruses (1, 3, 15, 20, 34, 38, 49, 53, 54). Us9 is a type II nonglycosylated membrane protein with no described biological activity apart from its role in neuronal transport (4, 18, 32). Here, we used several model systems to better characterize the roles of gE, gI, and Us9 in HSV-1 neuronal spread.Animal models to assess alphaherpesvirus neuronal transport (viral movement within a neuron) and spread (viral movement between cells) include the mouse flank and mouse retina models of infection. In the mouse flank model (Fig. ​(Fig.1A),1A), virus is scratch inoculated onto the depilated flank, where it infects the skin and spreads to innervating sensory neurons. The virus travels to the dorsal root ganglia (DRG) in the spinal cord (retrograde direction) and then returns to an entire dermatome of skin (anterograde spread). The virus also is transported in an anterograde direction from the DRG to the dorsal horn of the spinal cord and subsequently spreads to synaptically connected neurons. The production of zosteriform lesions and the presence of viral antigens in the dorsal horn of the spinal cord both are indicators of anterograde spread in this system. PRV gE and Us9 are required for the production of zosteriform disease, while gI is dispensable (7). In the absence of gE, HSV-1 also fails to cause zosteriform disease. However, unlike PRV, HSV-1 gE is required for retrograde spread to the DRG, so the role of gE in HSV-1 anterograde spread could not be evaluated in the mouse flank model (8, 36, 42).Open in a separate windowFIG. 1.Model systems to study HSV-1 neuronal spread. (A) Mouse flank model. Virus was scratch inoculated onto the skin, where it replicates, spreads to innervating neurons, and travels in a retrograde direction to the neuron cell body in the DRG. After replicating in the DRG, the virus travels in an anterograde direction back to the skin and into the dorsal horn of the spinal cord. Motor neurons also innervate the skin, allowing virus to reach the ventral horn of the spinal cord by retrograde transport. (B) Mouse retina model. Virus is injected into the vitreous body, from which it infects the retina as well as other structures of the eye, including the ciliary body, iris, and skeletal muscles of the orbit. From the retina, the virus is transported into the optic nerve and optic tract (OT) (anterograde direction) and then to the brain along visual pathways. Anterograde spread is detected in the lateral geniculate nucleus (LGN) and superior colliculus (SC). From the infected ciliary body, iris, and skeletal muscle, the virus spreads in a retrograde direction along motor and parasympathetic neurons and is detected in the oculomotor and Edinger-Westphal nuclei (OMN/EWN). Only first-order sites of spread to the brain are indicated. (Brain images were modified and reproduced from reference 47 with permission from of the publisher. Copyright Elsevier 1992.) (C) Campenot chamber system. Campenot chambers consist of a Teflon ring that divides the culture into three separate compartments. Neurons are seeded into the S chamber and extend their axons into the M and N chambers. Vero cells are seeded into the N chamber 1 day before infection. Virus is added to the S chamber and detected in the N chamber, a measure of anterograde spread.The mouse retina infection model (Fig. ​(Fig.1B)1B) has the advantage of allowing anterograde and retrograde spread to be studied independently of one another. Virus is delivered to the vitreous body, from which it infects the retina and other structures of the eye. The cell bodies of retinal neurons form the innermost layer of the retina; therefore, the virus infects these neurons directly, and spread from the retina along visual pathways to the brain occurs in an exclusively anterograde direction. In addition, the virus infects the anterior uveal layer of the eye (ciliary body and iris) and skeletal muscles in the orbit. From these tissues, the virus infects innervating parasympathetic and motor neurons and spreads to the brain in a retrograde direction. The localization of viral antigens in specific brain sites indicates whether the virus traveled to the brain along an anterograde or retrograde pathway (21, 25, 26, 39, 44, 51). PRV gE, gI, and Us9 each are essential for anterograde spread to the brain yet are dispensable for retrograde spread (5, 11, 25, 52). Even a strain of PRV lacking all three of these proteins retains retrograde neuronal spread activity (12, 40, 44). In contrast, in the absence of gE, HSV-1 fails to spread to the brain by either the anterograde or retrograde pathway (51).The Campenot chamber system (Fig. ​(Fig.1C)1C) has the advantage of allowing quantitative measurement of anterograde spread. Sympathetic neurons are cultured in a tripartite ring in which neuron cell bodies are contained in a separate compartment from their neurites. Virus is added to neuron cell bodies in one chamber, and anterograde spread to a separate chamber is measured by viral titers (13, 29, 30, 39, 43). Using this system, gEnull, gInull, and Us9null PRV each were shown to have only a partial defect in anterograde spread, while a virus lacking all three proteins was totally defective (13).We sought to quantify the anterograde spread activity of gEnull, gInull, and Us9null HSV-1 using the Campenot chamber system. While gEnull and gInull viruses were completely defective at anterograde spread, we found that a Us9null virus retained wild-type (WT) anterograde spread activity in this system. This observation was unexpected, since others previously had reported that Us9 is required for efficient HSV-1 anterograde transport or spread (26, 41, 46). Therefore, we further characterized the neuronal spread properties of two independent Us9-deleted viruses in the mouse retina and mouse flank models of infection. Our results indicate that gE and gI are essential for HSV-1 anterograde spread, whereas Us9 is dispensable.     

Genetic Addiction Risk Score (GARS): Molecular Neurogenetic Evidence for Predisposition to Reward Deficiency Syndrome (RDS)

We have published extensively on the neurogenetics of brain reward systems with reference to the genes related to dopaminergic function in particular. In 1996, we coined “Reward Deficiency Syndrome” (RDS), to portray behaviors found to have gene-based association with hypodopaminergic function. RDS as a useful concept has been embraced in many subsequent studies, to increase our understanding of Substance Use Disorder (SUD), addictions, and other obsessive, compulsive, and impulsive behaviors. Interestingly, albeit others, in one published study, we were able to describe lifetime RDS behaviors in a recovering addict (17 years sober) blindly by assessing resultant Genetic Addiction Risk Score (GARS?) data only. We hypothesize that genetic testing at an early age may be an effective preventive strategy to reduce or eliminate pathological substance and behavioral seeking activity. Here, we consider a select number of genes, their polymorphisms, and associated risks for RDS whereby, utilizing GWAS, there is evidence for convergence to reward candidate genes. The evidence presented serves as a plausible brain-print providing relevant genetic information that will reinforce targeted therapies, to improve recovery and prevent relapse on an individualized basis. The primary driver of RDS is a hypodopaminergic trait (genes) as well as epigenetic states (methylation and deacetylation on chromatin structure). We now have entered a new era in addiction medicine that embraces the neuroscience of addiction and RDS as a pathological condition in brain reward circuitry that calls for appropriate evidence-based therapy and early genetic diagnosis and that requires further intensive investigation.     

Evidence for a Role of the Epithelial Glycoprotein 40 (Ep-CAM) in Epithelial Cell-Cell Adhesion

Recently we have demonstrated that a 40kD human epithelium-specific glycoprotein exhibits the features of a homophilic cell-cell adhesion molecule, when expressed in transfected murine cells. We suggested the name Ep-CAM for this molecule (Litvinov et al., J. Cell Biol., 125: 437-446). Here we investigate the possible biological function of Ep-CAM in its natural environment—cells of epithelial origin. Immunolocalization of Ep-CAM in tissues and in cultures of epithelial/carcinoma cells showed that the majority of the Ep-CAM molecules are localized at cell-cell boundaries, predominantly along the whole lateral domain of polarized cells. In vitro, on single cells in suspension, the Ep-CAM molecules are present on the entire cell surface, and when the single cells grow attached, Ep-CAM is present at their pseudo-apical domain. During formation of intercellular contacts by such single cells, the majority of the Ep-CAM molecules are redistributed from the pseudoapical to the lateral domain of the cell membrane. Attachment of cells to the substrate does not cause redistribution of the molecules to the site of substrate attachment irrespective of the adhesive substrate (fibronectin, collagens, laminin, EHS-matrigel were tested). The monoclonal antibody 323/A3, reactive with the extracellular domain of the Ep-CAM molecule, has a strong negative effect on the aggregating behaviour of COV362 ovarian carcinoma cells and RC-6 immortalized mammary epithelial cells. The mAb affected cell aggregation in both cell lines in the presence of Ca⁺⁺, but with RC-6 cells the effect was more pronounced in low-calcium medium. The effects of the 323/A3 mAb on the already established intercellular contacts was not significant. The data presented demonstrate that the Ep-CAM molecules are functionally active in the epithelial and carcinoma cells tested, are capable of mediating Ca¹⁺-independent intercellular adhesions, and are not likely to be involved in cell-substrate adhesion.     

Evidence for a Role of the Epithelial Glycoprotein 40 (Ep-CAM) in Epithelial Cell-Cell Adhesion

Recently we have demonstrated that a 40kD human epithelium-specific glycoprotein exhibits the features of a homophilic cell-cell adhesion molecule, when expressed in transfected murine cells. We suggested the name Ep-CAM for this molecule (Litvinov et al., J. Cell Biol., 125: 437–446). Here we investigate the possible biological function of Ep-CAM in its natural environment—cells of epithelial origin. Immunolocalization of Ep-CAM in tissues and in cultures of epithelial/carcinoma cells showed that the majority of the Ep-CAM molecules are localized at cell-cell boundaries, predominantly along the whole lateral domain of polarized cells. In vitro, on single cells in suspension, the Ep-CAM molecules are present on the entire cell surface, and when the single cells grow attached, Ep-CAM is present at their pseudo-apical domain. During formation of intercellular contacts by such single cells, the majority of the Ep-CAM molecules are redistributed from the pseudoapical to the lateral domain of the cell membrane. Attachment of cells to the substrate does not cause redistribution of the molecules to the site of substrate attachment irrespective of the adhesive substrate (fibronectin, collagens, laminin, EHS-matrigel were tested). The monoclonal antibody 323/A3, reactive with the extracellular domain of the Ep-CAM molecule, has a strong negative effect on the aggregating behaviour of COV362 ovarian carcinoma cells and RC-6 immortalized mammary epithelial cells. The mAb affected cell aggregation in both cell lines in the presence of Ca⁺⁺, but with RC-6 cells the effect was more pronounced in low-calcium medium. The effects of the 323/A3 mAb on the already established intercellular contacts was not significant. The data presented demonstrate that the Ep-CAM molecules are functionally active in the epithelial and carcinoma cells tested, are capable of mediating Ca¹⁺-independent intercellular adhesions, and are not likely to be involved in cell-substrate adhesion.     

Ⅰ型单纯疱疹病毒 gD基因片段的高效表达及抗原性分析

目的 获得高表达的Ⅰ型单纯疱疹病毒(HSV)被膜糖蛋白gD（简称gD1）基因的工程菌。方法 通过计算机分析,筛选出疱疹病毒gD1中优势抗原决定簇的基因片段。将克隆的基因片段插入表达载体pTrxA内,转化大肠杆菌Rosetta,以异丙基-β-D-硫代半乳糖苷诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。　结果　PCR扩增出约930bp的gD1编码基因目的片段,与预期片段大小相符,经测序鉴定无基因突变;所构建pTrxA-gD1重组表达质粒阳性克隆经PCR与双酶切鉴定,与预期结果一致;含有pTrxA-gD1重组质粒的大肠杆菌Rosetta诱导后得到了高效达,SDS-PAGE显示表达产物约Mr48000（Dalton）。免疫印迹结果表明表达产物具有较好的抗原性。结论　成功构建了pTrxA-gD1表达质粒,实现了成熟gD1蛋白在大肠杆菌中的高效表达,表达产物具有好的抗原性。     

Evidence for Genetic Factors Underlying the Insulin Resistance Syndrome in American Indians

Previous analyses of American Indians of the Strong Heart Study have demonstrated that various insulin‐resistance variables cluster, although knowledge about the genetic determination of these clusters is unknown. In an effort to explore the influences on the aggregation of insulin‐resistance phenotypes, we used principal component factor analysis to examine the clustering of these phenotypes in participants of the Strong Heart Family Study and evaluated the genetic and environmental contributions of such factors. Nine traits were chosen for principal component factor analysis: BMI, diastolic blood pressure, fasting glucose, high‐density lipoprotein‐cholesterol, natural log‐transformed insulin, natural log‐transformed triglycerides, percentage of body fat, systolic blood pressure, and waist‐to‐hip ratio. Analyses revealed three clusters: glucose/insulin/obesity, blood pressure, and dyslipidemia factors. Using a variance component approach and accounting for the effects of age, sex, center, and medication, we detected significant heritabilities (h²) for the three factors: h² = 0.67, h² = 0.33, and h² = 0.61, respectively. In multivariate analysis, no significant genetic correlations among factors were found. These results suggest that heredity explains a substantial proportion of the variability of the factors that underlie the insulin resistance syndrome in American Indians and that these factors are genetically independent.     

Evidence for Genetic Change in the Antirrhinum Rust (Puccinia antirrhini)

The available data on the resistance of Antirrhinum majus L.to rust, Puccinia antirrhini Dietel and Holway, are reviewed.There is evidence for one or more genetic changes in the pathogen.In one instance it can be demonstrated that this change is likelyto have been by mutation. The chronology of the changes is:in the U.S.A., east of the Rockies between 1921 and 1935 andin California during 1936; in Europe between 1936 and 1954 andin Britain between 1958 and 1962. A virulent race was presentin Australia by 1954. Puccinia antirrhini Dietel and Holway, rust, Antirrhinum majus L., mutation, virulence, resistance     

A Multiparametric Computational Algorithm for Comprehensive Assessment of Genetic Mutations in Mucopolysaccharidosis Type IIIA (Sanfilippo Syndrome)

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo syndrome) is a Lysosomal Storage Disease caused by cellular deficiency of N-sulfoglucosamine sulfohydrolase (SGSH). Given the large heterogeneity of genetic mutations responsible for the disease, a comprehensive understanding of the mechanisms by which these mutations affect enzyme function is needed to guide effective therapies. We developed a multiparametric computational algorithm to assess how patient genetic mutations in SGSH affect overall enzyme biogenesis, stability, and function. 107 patient mutations for the SGSH gene were obtained from the Human Gene Mutation Database representing all of the clinical mutations documented for Sanfilippo syndrome. We assessed each mutation individually using ten distinct parameters to give a comprehensive predictive score of the stability and misfolding capacity of the SGSH enzyme resulting from each of these mutations. The predictive score generated by our multiparametric algorithm yielded a standardized quantitative assessment of the severity of a given SGSH genetic mutation toward overall enzyme activity. Application of our algorithm has identified SGSH mutations in which enzymatic malfunction of the gene product is specifically due to impairments in protein folding. These scores provide an assessment of the degree to which a particular mutation could be treated using approaches such as chaperone therapies. Our multiparametric protein biogenesis algorithm advances a key understanding in the overall biochemical mechanism underlying Sanfilippo syndrome. Importantly, the design of our multiparametric algorithm can be tailored to many other diseases of genetic heterogeneity for which protein misfolding phenotypes may constitute a major component of disease manifestation.     

Heterogeneity in photosystem I. Evidence from cation-induced decrease in Photosystem I electron transport

The rate of electron transfer through Photosystem I (reduced 2,6-dichlorophenol indophenol (DCIPH₂ → methylviologen) in a low-salt thylakoid suspension is inhibited by Mg²⁺ both under light-limited and the light-saturated conditions, the magnitude of inhibition being the same. The 2,6-dichlorophenol indophenol (DCIP) concentration dependence of the light-saturated rate in the presence and in the absence of Mg²⁺ shows that the overall rate constant of the photoreaction is not altered by Mg²⁺. With N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethylphenylenediamine as electron donor only the light-limited rate, not the light-saturated rate, is inhibited by Mg²⁺ and the magnitude of inhibition is the same as with DCIP as donor. The results are interpreted in terms of heterogeneous Photosystem I, consisting of two types, PS I-A and PS I-B, where PS I-A is involved in cation-regulation of excitation energy distribution and becomes unavailable for DCIPH₂ → methyl viologen photoelectron transfer in the presence of Mg²⁺.     

Genetic Analysis of the Role of Herpes Simplex Virus Type 1 Glycoprotein K in Infectious Virus Production and Egress

Herpes simplex virus type 1 (KOS)DeltagK is a mutant virus which lacks glycoprotein K (gK) and exhibits defects in virion egress (S. Jayachandra, A. Baghian, and K. G. Kousoulas, J. Virol. 69:5401-5413, 1997). To further understand the role of gK in virus egress, we constructed recombinant viruses, DeltagKhpd-1, -2, -3, and -4, that specified gK amino-terminal portions of 139, 239, 268, and 326 amino acids, respectively, corresponding to truncations immediately after each of the four putative membrane-spanning domains of gK. DeltagKhpd-1 and DeltagKhpd-2 viruses produced lower yields and smaller plaques than DeltagK. Numerous DeltagKhpd-1 capsids accumulated predominately within large double-membrane vesicles of which the inner membrane appeared to be derived from viral envelopes while the outer membrane appeared to originate from the outer nuclear membrane. The mutant virus DeltagKhpd-3 produced higher yields and larger plaques than the DeltagK virus. The mutant virus DeltagKhpd-4 produced yields and plaques similar to those of the wild-type virus strain KOS, indicating that deletion of the carboxy-terminal 12 amino acids did not adversely affect virus replication and egress. Comparisons of the gK primary sequences specified by alphaherpesviruses revealed the presence of a cysteine-rich motif (CXXCC), located within domain III in the lumen side of gK, and a tyrosine-based motif, YTKPhi (where Phi is any bulky hydrophobic amino acid), located between the second and third hydrophobic domains (domain II) in the cytoplasmic side of gK. The mutant virus gK/Y183S, which was constructed to specify gK with a single-amino-acid change (Y to S) within the YTKPhi motif, replicated less efficiently than the DeltagK virus. The mutant virus gK/C304S-C307S, which was constructed to specify two serine instead of cysteine residues within the cysteine-rich motif (CXXCC changed to SXXSC) of gK domain III, replicated more efficiently than the DeltagK virus. Our data suggests that gK contains domains in its amino-terminal portion that promote aberrant nucleocapsid envelopment and/or membrane fusion between different virion envelopes and contains domains within its domains II and III that function in virus replication and egress.     

Heterogeneity of the internal transcribed spacer 1 (ITS1) inTulipa (Liliaceae)

Heterogeneity of the internal transcribed spacer ITS1 of the rDNA within individuals ofTulipa gesneriana L.,T. kaufmanniana Regel, and their interspecific hybrids was analyzed by PCRRFLP, using the polymorphic restriction enzymesRsaI andHinfI, and by nucleotide sequence analysis. In most cases, the sum of the sizes of the restriction fragments was higher than the entire length of the undigested ITS fragment, indicating heterogeneity at the restriction sites within an individual. Differences in band intensities within the restriction patterns indicate the occurrence of variation in copy number of these different ITS1 variants within individuals. Automated sequencing without a visual inspection often failed to detect existing heterogeneity within sequences, resulting in a discrepancy between the sequencing and restriction analysis results. By visual interpretation of the sequences, the restriction patterns could mostly be predicted well. Fluorescence in situ hybridization (FISH) experiments in fourTulipa species revealed the occurrence of several rDNA spots. The number of rDNA loci varied from seven inT. gesneriana Christmas Marvel to ten inT. australis Link. This might explain the occurrence of heterogeneity in ITS sequences inTulipa, as homogenization of variants has to take place over different loci.     

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.     

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.