The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA.

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278-283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246-259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.

U-73122: a potent inhibitor of human polymorphonuclear neutrophil adhesion on biological surfaces and adhesion-related effector functions.

We have reported that U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole- 2,5-dione) an inhibitor of phospholipase C-dependent processes in human polymorphonuclear neutrophils (PMN) and platelets, potently suppresses the responsiveness of suspended PMN and platelets to receptor agonists. We demonstrate here that U-73122 caused a concentration-dependent (10-800 nM) inhibition of N-formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor-alpha (TNF alpha), interleukin-8 and phorbol myristate acetate (PMA)-triggered PMN adhesion on fibronectin, fetal bovine serum or keyhole limpet hemocyanincoated microtiter plates. U-73122 also inhibited PMN adherence to and transmigration through TNF-alpha-activated endothelium (IC50 < 50 nM). Further, U-73122 suppressed interleukin-8, N-formylmethionyl-leucyl-phenylalanine and PMA-stimulated up-regulation of the beta 2-integrin, Mac-1 (CD11b/CD18), on the PMN surface (IC50 < 1.3 microM). U-73122 also caused a time-(15-120 min) and concentration-dependent inhibition (IC50 = 25-100 nM) of the N-formyl-methionyl-leucyl-phenylalanine-, TNF alpha- and PMA-elicited adhesion-dependent, oxidative burst, measured as hydrogen peroxide (H2O2) production, in PMN. The CD18-dependent extracellular release of lactoferrin from PMN activated with these stimuli was also suppressed by U-73122. U-73343 (1-[6-[[17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine dione), a close analog of U-73122, did not affect PMN responsiveness.

High-sulfur protein gene expression in a transgenic mouse.

We analyzed the effect of minoxidil on hair follicles isolated from transgenic mice. These transgenic animals synthesize the reporter enzyme CAT in their hair follicles only during the active phases of hair growth. The recombinant gene used to generate these mice contained the bacterial enzyme CAT under the control of the promoter from the gene of UHS protein. Studies using in situ hybridization showed that UHS proteins are expressed specifically in the matrix cells of the hair follicle during the terminal stages of hair differentiation. Hence the expression of the UHS proteins is a clear sign of active hair growth. With other in situ hybridization studies we demonstrated that CAT mRNA is expressed in differentiating matrix cells of the hair shaft in a location similar to that in which mRNA encodes UHS proteins. Thus we can use the levels of CAT activity as a measure of hair growth. We have confirmed that expression of the transgene is found in hair that is high in anagen and low in catagen follicles. The usefulness of our model was further demonstrated by showing that minoxidil, a drug that stimulates hair growth, increased the expression of CAT in cultured hair follicles. Thus we have demonstrated that expression of this reporter gene is sensitive, hair specific, and also useful for monitoring effects in cultured hair follicles. Hence these transgenic mice provide a model system for studying the biology of hair growth.

Hair-specific keratins: characterization and expression of a mouse type I keratin gene.

A genomic clone for a member of the mouse type I hair keratin protein family has been isolated and analyzed in order to study the regulation of this keratin during the hair growth cycle. The coding sequence is divided into seven exons. The gene structure is typical of keratins in particular and intermediate filaments in general in that the intron-exon borders are not located at the domain borders of the protein. Comparison with a sheep wool keratin gene shows that the splice sites in the two hair keratin genes are found at identical locations relative to the amino acid sequence of the proteins. Similarly, comparison of the promoter areas of these genes shows several areas of nucleotide sequence conservation, including the area around the TATA box and an SV40 core enhancer sequence. In addition, a high degree of sequence identity exists in the fourth intron. In situ hybridization shows that transcripts of this gene are first found in the relatively undifferentiated proximal cortex area in the keratogenous zone of mouse vibrissae.

Hair-specific expression of chloramphenicol acetyltransferase in transgenic mice under the control of an ultra-high-sulfur keratin promoter.

We have generated a transgenic mouse line by microinjection of a chimeric DNA fragment (KER-CAT) containing a hair-specific, murine ultra-high-sulfur keratin promoter (KER) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. A 671-base pair (bp) stretch of the 5' promoter region was used to direct the expression of the CAT gene in this construct. Of the tissues tested for CAT activity in these transgenic animals only skin with growing hair, isolated hair follicles, and microdissected vibrissae showed substantial levels of activity. These are the same tissues where the endogenous ultra-high-sulfur keratin gene is expressed as shown by in situ hybridization. Furthermore, analysis of the CAT activity during the developmental stages of the hair growth cycle shows that the chimeric gene is expressed during the anagen phase of the hair growth cycle; this is the expected time during development for its expression. From these results we conclude that 671 bp of the promoter sequence from the ultra-high-sulfur keratin gene is sufficient to direct the correct development-specific and tissue-specific expression of the reporter gene construct in transgenic mice. The appropriate expression of the KER-CAT construct in transgenic mice is an important step in understanding the regulation of this gene during hair organogenesis.

An ultra-high sulfur keratin gene is expressed specifically during hair growth.

To study the regulation of the hair cycle in the mouse, we have isolated and characterized a gene for ultra high sulfur keratin that is expressed specifically during the active hair growth cycle. The gene (gUHSK-704Eco) was isolated as a member of a gene cluster on a recombinant phage with a DNA insert of 18 kb that was isolated by screening a murine genomic library at low stringency with a synthetic oligonucleotide derived from a sheep high sulfur keratin gene (Powell, Nucleic Acids Res. 1983 11, 5327). The murine ultra-high sulfur keratin gene has no intervening sequence; the 558 nucleotide of the coding region specify 186 amino acids, of which 70 (37%) are cysteine. A Cys-Cys-Gln-Pro repeat is found 12 times within the coding region. RNA dot blots show that the ultra-high sulfur keratin gene is expressed during the hair cycle concomitant with the anterior-posterior temporal pattern of the normal murine hair cycle.

Nuclear precursor molecules of the two beta-globin mRNAs in Friend erythroleukemia cells.

Processing of the beta major and beta minor globin pre-mRNAs has been compared in murine erythroleukemia cells induced to synthesize hemoglobin by dimethyl sulfoxide or hemin treatment, using both the Northern blot technique and S1 nuclease mapping with 3' and 5' end-labeled probes. The small intervening sequence of both beta-globin pre-mRNAs was removed in one step, although minor amounts of incompletely spliced RNA were detected. During the processing of the large intervening sequence of beta major globin pre-mRNA two internal splice sites were clearly detected. On the contrary, the beta minor globin pre-mRNA did not show any internal splice sites. A model of processing of the mouse adult beta major globin pre-mRNA is proposed.