Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the U L 34 gene cannot undergo this event. U L 34 and U L 31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of U L 34 that are necessary for primary envelopment to occur, a library of 19 U L 34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (⌬TM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Herpes simplex virus type 1 (HSV-1) undergoes primary envelopment, budding from the interior of the nucleus into the perinuclear space, as one of the first steps in egress from the cell (10,29,34). Herpesvirus egress is a complex process involving many virus-encoded proteins (2-4, 12, 13, 18, 21, 26), but only the U L 34 protein and its homologs have been shown to be required for primary envelopment in HSV-1 (35), pseudorabies virus (22), herpes simplex virus type 2 (HSV-2) (39), and most recently in equine herpesvirus type 1 (28). The HSV-1 U L 34 gene product is a 30-kDa membrane-associated phosphoprotein that is a substrate for the virus-encoded protein kinase U S 3 (31, 32). The U L 34 sequence contains a putative transmembrane domain of 14 to 17 amino acids near the C terminus of the protein. While localization and membrane association of the U L 34 protein suggest that U L 34 contains a transmembrane anchor, biochemical tests have not been done to verify the existence of such a domain. In addition, the interaction of U L 34 with another HSV-1 protein, the U L 31 gene product, is necessary and sufficient to target both proteins to the nuclear membrane, where they are thought to form a complex (33). Colocalization of U L 34 and U L 31 at the nuclear membrane is also seen in HSV-2 and pseudorabies virus (16,43). Since the U L 34 protein coding sequence is well conserved in alphaherpesviruses for which sequences are available (11,15,40), it seems likely that U L 34 protein function is also widely conserved among herpesviruses.Efficient primary envelopment in alphaherpesviruses is linked to complete packaging of DNA into the capsid. Many viral mutants that synthesize capsids but fail to correctly package their DNA are unable to envelop the empty capsids (1,6,7,19,25,30,36). Once genome packaging is complete, the nucleocapsid must gain access to the inner nuclear membrane, and this, in turn, likely requires a mechanism to break down the nuclear lamina. It is currently unknown how HSV-1 gains access to the nuclear membrane, but several lines of evidence suggest that herpesvirus infection leads to alterations of nuclear envelope structure. Infection with HSV-1 has been reported to lead to an increase in soluble lamin A/C, suggesting at least limited disassembly of the lamina (38)...

The metF gene in Escherichia coli and Salmonella typhimurium is under negative transcriptional control by the MetJ repressor. Expression of an S. typhimurium metF-lacZ gene fusion is repressed up to 10-fold by methionine addition to the growth medium in E. coli hosts encoding wild-type MetJ repressor; this repression is not seen in metJ mutants. metR mutations which eliminate the MetR activator protein result in two-to threefold-more-severe repression by the MetJ repressor. In a metJ metR double mutant, however, the level of metF-lacZ expression is the same as in a metj mutant, suggesting that MetR antagonizes MetJ-mediated methionine repression of the metF promoter. A DNA footprint analysis showed that MetR binds to a DNA fragment carrying the metF promoter and protects two separate regions from DNase I digestion: a 46-bp region from position -50 to -95 upstream of the transcription initiation site and a 24-bp region from about position +62 to +85 downstream of the transcription initiation site and within the metF structural gene. Nucleotide changes in each of the MetR-binding sites away from the consensus sequence disrupt MetR-mediated regulation of the metF-lacZ fusion.The folate branch of the methionine pathway in Salmonella typhimunum includes the metE, metH, and metF genes. The metF gene product (5,10-methylenetetrahydrofolate reductase) is involved in the biosynthesis of 5-methyltetrahydrofolate from 5,10-methylenetetrahydrofolate (20 (24), and pFlac carries a fusion of the S. typhimunum metF control region and the first 12 amino acid codons of the structural gene to the eighth codon of lacZ (25).Media and growth conditions. Luria agar, Luria broth, and glucose minimal medium (GM) have been described previously (23). Supplements were added at the following concentrations: amino acids, 50 ,ug/ml; vitamin B1, 1 ,ug/ml; ampicillin, 150 pug/ml; and 5-bromo-4-chloro-3-indolyl-P-Dgalactopyranoside (X-Gal), 40 ,ug/ml. 13-Galactosidase enzyme assay. ,B-Galactosidase activity was assayed as described by Miller (17), using the chloroform-sodium dodecyl sulfate lysis procedure. Results are the averages of two or more assays in which each sample was measured in triplicate.DNA manipulation. Restriction enzyme digestions, ligations, plasmid and phage DNA isolations, and DNA fragment isolations were done as described by Maniatis et al. (14). Transformations were done by using polyethylene glycol-and dimethyl sulfoxide-prepared competent cells (5).Site-directed mutagenesis. A 746-bp EcoRI-BamHI fragment carrying the metF control region was isolated from plasmid pFlac (25)

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A 3-T study is presented, comparing the ability of two 1 H spectroscopy pulse sequences, Carr-Purcell point resolved spectroscopy (CPRESS; TE 5 45 msec), and conventional PRESS (TE 5 35 msec), to separate between groups of 20 normal control (NC) and 20 mild cognitive impairment (MCI) subjects. Both sequences showed higher myo-inositol (mI) and mI/N-acetyl-aspartate (NAA) levels in the posterior cingulate gyrus of the MCI subjects. The increased intrasubject repeatability of mI and mI/NAA CPRESS measurements (~6% vs. 9% for PRESS) translated into decreased intraclass variability. A 22% intraclass mI PRESS variability was reduced to 16% for CPRESS, and an 18% intraclass mI/NAA PRESS variability was reduced to 12% for CPRESS for the group of NC subjects. Similar results were observed for the MCI subjects. Decreased intraclass variability led to improved separation between NC and MCI subjects (P 5 0.017 for PRESS and P < 0.0001 for CPRESS mI/NAA, the best NC/MCI discriminant for each method). Seventy-five percent sensitivity at eighty percent specificity was demonstrated by mI/NAA CPRESS measurements in separating NC from MCI subjects. High correlations were also observed between subject performance on a number of neuropsychological tests (probing verbal memory, visuoconstruction performance, and visual motor integration) and the mI/NAA ratio; higher correlation coefficients (with stronger statistical significance) were consistently evident for CPRESS than for PRESS data. Magn Reson Med 65:1515-1521,