Beta-galactosidase Enzyme Activity Research Articles

An optimised protocol for the detection of lipofuscin, a versatile and quantifiable marker of cellular senescence.

Lipofuscin is a yellow-brown pigment typically found in the lysosomes that contains a mixture of molecules including lipids, metals and misfolded proteins. The use of Sudan black B to detect lipofuscin accumulation, a well described marker of cellular senescence and ageing, was first described in 2013 by Georgakopoulou, et al. Here, we provide an optimisation of the original protocol. Firstly, we adjusted the staining methodology for increased ease of use on cultured cells. Secondly, we show that Sudan black B-stained lipofuscin emits strong fluorescence in the far-red channel making it suitable for fluorescence microscopy detection and quantification. Moreover, we also demonstrate that this optimised protocol can be utilised in conjunction with standard immunofluorescence staining techniques, making possible the simultaneous detection of lipofuscin and other cellular proteins of interest, like additional markers of senescence. This is a significant advantage over the most commonly used method for senescence detection, based on beta galactosidase enzymatic activity. We therefore believe that these findings and the provided optimised protocol will represent a useful tool for the scientific community in the field of cellular senescence.

The covalent immobilization of β-galactosidase from Aspergillus oryzae and alkaline protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes in milk

This study aimed was to covalently immobilize β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes. In this study, a two-level factorial design was employed to investigate the impact of seven continuous variables (activation pH, glutaraldehyde molarity, activation time (0–8 h), buffer solution pH (8-0), buffer solution molarity, MWCNT-NH2-glutaraldehyde quantity, and stabilization time (0–180 h)) on the immobilization efficiency and enzymatic activity of protease and β-galactosidase. Furthermore, the effect of time on the percentage of enzymatic activity was examined during specific intervals (24, 48, 72, 96, and 120 h) of the immobilization process. The analysis of variance results for protease enzymatic activity revealed a notable influence of the seven variables on immobilization efficiency and enzymatic activity. Additionally, the findings indicate that activation time, buffer pH, MWCNT-NH2-glutaraldehyde quantity, and stabilization time significantly affect the activity of the protease enzyme. The interplay between buffer pH and stabilization time is also significant. Indeed, both activation time and the quantity of MWCNT-NH2-glutaraldehyde exert a reducing effect on enzyme activity. Notably, the influence of MWCNT-NH2-glutaraldehyde quantity is more significant (p < 0.05). In terms of beta-galactosidase enzymatic activity, the study results highlight that among the seven variables considered, only the glutaraldehyde molarity, activation time, and the interplay of activation time and the quantity of MWCNT-NH2-glutaraldehyde can exert a statistically significant positive impact on the enzyme's activity (p < 0.05). The combination of activation time and buffer solution molarity, as well as the interactive effect of buffer pH and MWCNT–NH2–glutaraldehyde, can lead to a significant improvement in the stabilization efficiency of the protease of carbon nanotubes. The analysis of variance results demonstrated that the efficiency of covalently immobilizing β-galactosidase from Aspergillus oryzae on amino-functionalized multi-walled carbon nanotubes is influenced by the molarity of glutaraldehyde, buffer pH, stabilization time, and the interplay of activation time + buffer pH, buffer pH + activation time, activation time + buffer molarity, and glutaraldehyde molarity + MWCNT-NH2-glutaraldehyde (p < 0.05). Through the optimization and selection of optimal formulations, the obtained results indicate enzyme activities and stabilization efficiencies of 64.09 % ± 72.63 % and 65.96 % ± 71.77 % for protease and beta-galactosidase, respectively. Moreover, increasing the enzyme stabilization time resulted in a reduction of enzyme activity. Furthermore, an increase in pH, temperature, and the duration of milk storage passing through the enzyme-immobilized carbon nanotubes led to a decrease in enzyme stabilization efficiency, and lactose hydrolysis declined progressively over 8-h. Hence, the covalent immobilization of β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis onto amino-functionalized multi-walled carbon nanotubes is anticipated to be achievable for milk applications.

Single Institutional Experience with GM1 Gangliosidosis: Clinical and Laboratory Results of 14 Patients

Background:GM1 gangliosidosis is an autosomal recessive lysosomal storage disease caused by biallelic mutations in the GLB1 gene. Neurodegeneration, hypotonia, visceromegaly, macular cherry-red spots, skeletal dysplasia, and coarse and dysmorphic face are the major clinical features.Aims:To evaluate the demographic and clinical data of patients with GM1 gangliosidosis in a single center.Study Design:A retrospective clinical study.Methods:This study included patients followed at Hacettepe University İhsan Doğramacı Children’s Hospital Pediatric Metabolism Unit with the diagnosis of GM1 gangliosidosis between 1988 and 2021. Hospital records of the patients were reviewed for demographic, clinical, and laboratory findings.Results:Fourteen patients were included in the study and 10 (71.4%) were male. The age at onset of clinical symptoms was between 0 and 5 months, and the median time to diagnosis after the first symptom was 4.3 (0-13) months. Motor delay (54%) was the most common initial symptom. The median follow-up period was 14.8 (0.4-92.2) months. Twelve patients (85.7%) died, and all deaths occurred before the age of 24 months. The median survival was 21.3 (95% confidence interval, 15.5-24.9) months. Higher leukocyte beta-galactosidase activity correlated with later age at onset (ρ = 0.575), later age at diagnosis (ρ = 0.618), and longer diagnostic delay (ρ = 0.702) (ρ < 0.05).Conclusion:Median survival in patients with GM1 gangliosidosis is less than 24 months. Beta-galactosidase enzyme activity may be associated with clinical onset and time of diagnosis in these patients.

Comparable in vitro Function of Human Liver-Derived and Adipose Tissue-Derived Mesenchymal Stromal Cells: Implications for Cell-Based Therapy.

Mesenchymal stem/stromal cells (MSCs) have been investigated extensively for their immunotherapeutic and regenerative properties, which may differ by cell source. In MSCs harvested from donors matched for sex, age, and body mass index, we compared the proliferative and migration functions of liver-derived MSCs (L-MSCs) and adipose tissue-derived MSCs (A-MSCs) (n = 6 donors each). Cellular senescence was evaluated by senescence-associated beta-galactosidase enzyme activity and expression of senescence-associated secretory phenotype (SASP) factors using real-time quantitative polymerase chain and by western blot assay. The pro-angiogenic and reparative potency of MSCs was compared by co-culturing MSCs with injured human umbilical vein endothelial cells (HUVEC). The proliferation and migration properties were similar in L-MSCs and A-MSCs. Although cell cycle arrest and SASP genes were similarly expressed in both MSCs, tumor necrosis factor alpha gene and protein expression were significantly downregulated in L-MSCs. In co-cultured injured HUVEC, A-MSCs restored significantly more tubes and tube connections than L-MSCs. Therefore, despite many functional similarities between L-MSCs and A-MSCs, L-MSCs have enhanced immunomodulatory properties, while A-MSCs appear to have better pro-angiogenic and vascular reparative potency. Availability of a broad range of cellular options might enable selecting cell-based therapy appropriate for the specific underlying disease.

Determination of beta-galactosidase enzyme activities of Lactobacillus strains

Determination of beta-galactosidase enzyme activities of Lactobacillus strains

Conformational behaviour determines the low-relaxivity state of a conditional MRI contrast agent

The conformational behaviour in aqueous solution of the EgadMe complex, a conditional gadolinium-based contrast agent sensitive to beta-galactosidase enzymatic activity, is investigated by means of ab initio calculations and classical molecular dynamics simulations. Furthermore, force field parameterization of gadolinium-ligand interactions is performed, and its reliability is tested on the bench mark [Gd(DOTA)](-) system by MD simulations. Both computational methods highlight the presence in EgadMe of two main conformational isomers. The lowest energy conformation is a "close" form, corresponding to a state of low-relaxivity (MRI "inactive"), in which the ninth coordination site of the gadolinium ion is occupied by one oxygen atom of the galactopyranose residue. The second isomer, which is 2.9 (at ab initio level) and 4.2 (at MD level) kcal mol(-1) above the global minimum, presents an "open" form, corresponding to a state of high-relaxivity (MRI "active") in which one water molecule coordinates the ion. These results are consistent with experimental findings reported for EgadMe, and show that competition at the ninth coordination site of gadolinium ion, between the intra (the galactopyranose residue) and inter (water molecules) molecular interactions, affects the relaxivity of this system.

Microarray analysis of developing flax hypocotyls identifies novel transcripts correlated with specific stages of phloem fibre differentiation.

Hypocotyls are a commonly used model to study primary growth in plants, since post-germinative hypocotyls increase in size by cell elongation rather than cell division. Flax hypocotyls produce phloem fibres in bundles one to two cell layers thick, parallel to the protoxylem poles of the stele. Cell wall deposition within these cells occurs rapidly at a well-defined stage of development. The aim was to identify transcripts associated with distinct stages of hypocotyl and phloem fibre development. Stages of flax hypocotyl development were defined by analysing hypocotyl length in relation to fibre secondary wall deposition. Selected stages of development were used in microarray analyses to identify transcripts involved in the transition from elongation to secondary cell wall deposition in fibres. Expression of specific genes was confirmed by qRT-PCR and by enzymatic assays. Genes enriched in the elongation phase included transcripts related to cell-wall modification or primary-wall deposition. Transcripts specifically enriched at the transition between elongation and secondary wall deposition included beta-galactosidase and arabinogalactan proteins. Later stages of wall development showed an increase in secondary metabolism-related transcripts, chitinases and glycosyl hydrolases including KORRIGAN. Microarray analysis also identified groups of transcription factors enriched at one or more stages of fibre development. Subsequent analysis of a differentially expressed beta-galactosidase confirmed that the post-elongation increase in beta-galactosidase enzyme activity was localized to phloem fibres. Transcripts were identified associated with specific stages of hypocotyl development, in which phloem fibre cells were undergoing thickening of secondary walls. Temporal and spatial regulation of beta-galactosidase activity suggests a role for this enzyme in remodelling of flax bast fibre cell walls during secondary cell wall deposition.

Characterization and prevalence of Solobacterium moorei associated with oral halitosis

Previous studies suggest that Solobacterium moorei is associated with oral halitosis. In the present study, we examined the prevalence of S. moorei on the dorsal surface of the tongue in 57 adults (21 with and 36 without halitosis) by bacterial culture and direct amplification of nucleic acids. We also examined the S. moorei type strain and four clinical isolates for 16S ribosomal nucleic acid sequence, H2S and enzyme production, and antibiotic susceptibility. S. moorei was found on the dorsal surface of the tongue in 100% of the subjects with halitosis and 14% of subjects without halitosis. Infection with S. moorei was correlated with organoleptic measures of halitosis and with volatile sulfur compound levels. Nucleic acid probe detection of S. moorei as a test for halitosis exhibited 100% sensitivity and 86% specificity. The S. moorei type strain and all four clinical isolates showed >98% 16S rDNA sequence similarity, produced H2S, demonstrated acid phosphatase, beta-galactosidase, alpha-glucosidase, esterase, leucine arylamidase and naphthol phosphohydrolase enzyme activities, and were sensitive to all antibiotics tested except gentamicin, kanamycin, nalidixic acid and rifampin. This study supports the hypothesis that S. moorei is associated with halitosis.

Intrauterine growth retardation and placental vacuolization as presenting features in a case of GM1 gangliosidosis

Diagnosis of GM1 gangliosidosis (OMIM 230500) is usually based on the presence of physical signs of storage such as coarse facial features, corneal clouding, cherry red macula, hepatosplenomegaly and skeletal dysostosis. More rarely it can present as nonimmune hydrops. We describe a male patient with GM1 gangliosidosis born to healthy first-cousin parents of Indian Asian descent. The disease was recognized on the basis of diffuse vacuolization of cyto- and syncytiotrophoblasts, stromal cells and amniocytes on histological analysis of the placenta. The placental examination was prompted by the prenatal detection of intrauterine growth retardation (IUGR) and oligohydramnios at 32 weeks of gestation. The diagnosis of GM1 gangliosidosis was supported by both biochemical and molecular data. The beta-galactosidase enzymatic activity on leukocytes was severely reduced, while the neuraminidase activity on fibroblasts was normal, thereby excluding galactosialidosis. The molecular analysis of the beta-galactosidase gene (GLB1) revealed a previously unreported splicing mutation (IVS1+2 insT) in homozygous state. Our case further illustrates the value of histological examination of the placenta in the diagnosis of lysosomal storage disorders and shows that either hydrops or IUGR can be presenting features of GM1 gangliosidosis in the neonatal period.

Precise excision of long terminal repeats of the gypsy (mdg4) retrotransposon of Drosophila melanogaster detected in Escherichia coli cells is explained by its integrase function

An Escherichia coli model system was developed to estimate the capacity of the integrase of the Drosophila melanogaster retrotransposon gypsy (mdg4) for precise excision of the long terminal repeat (LTR) and, hence, the entire gypsy. The gypsy retrotransposon was cloned in the form of a PCR fragment in the pBlueScript II KS+ (pBSLTR) vector, and the region of the second open reading frame (INT ORF2) of this element encoding integrase was cloned under the lacZ promoter in the pUC19 vector and then recloned in pACYC184 compatible with pBSLTR. The LTR was cloned in such a manner that its precise excision from the recombinant plasmid led to the restoration of the nucleotide sequence and the function of the ORF of the lacZ gene contained in the vector; therefore, it was detected by the appearance of blue colonies on a medium containing X-gal upon IPTG induction. Upon IPTG induction of E. coli XL-1 Blue cells obtained by cotransformation with plasmids pACCint and pBSLTR on an X-gal-containing medium, blue clones appeared with a frequency of 1 x 10(-3) to 1 x 10(-4), the frequency of spontaneously appearing blue colonies not exceeding 10(-9) to 10(-8). The presence of blue colonies indicated that that the integrase encoded by the INT ORF2 (pACYC 184) fragment was active. After the expression of the integrase, it recognized and excised the gypsy LTR from pBSLTR, precisely restoring the nucleotide sequence and the function of the lacZ gene, which led to the expression of the beta-galactosidase enzymatic activity. PCR analysis confirmed that the LTR was excised precisely. Thus, the resultant biplasmid model system allowed precise excisions of the gypsy LTR from the target site to be detected. Apparently, the gypsy integrase affected not only the LTR of this mobile element, but also the host genome nucleotide sequences. The system is likely to have detected only some of the events occurring in E. coli cells. Thus, the integrase of gypsy is actually capable of not only transposing this element by inserting DNA copies of the gypsy retrotransposon to chromosomes of Drosophila, but also excising them, gypsy is excised via a precise mechanism, with the original nucleotide sequence of the target site being completely restored. The obtained data demonstrate the existence of alternative ways of the transposition of retrotransposons and, possibly, retroviruses, including gypsy (mdg4).

Chronic Pseudomonas aeruginosa infection reduces surfactant levels by inhibiting its biosynthesis

Chronic Pseudomonas aeruginosa infection, as occurs in cystic fibrosis, is associated with decreased surfactant phospholipid levels. To investigate mechanisms, we measured synthesis of dipalmitoylphosphatidylcholine (DPPC), the major surfactant phospholipid. Mice received an agarose bead slurry alone, or were infected with beads containing a clinical mucoid isolate of P. aeruginosa. Bacterial infection after 3 days resulted in a approximately 50% reduction in surfactant DPPC content versus control. These changes in surfactant were associated with co-ordinate reductions in mRNAs and immunoreactive levels for CTP: phosphocholine cytidylyltransferase (CCTalpha), the rate-regulatory enzyme required for DPPC synthesis. P. aeruginosa infection of murine lung epithelia decreased CCTalpha gene transcription without altering mRNA stability and by a mechanism other than release of a soluble extracellular inhibitor. Promoter deletional analysis revealed that P. aeruginosa activates a negative response element from -1019 to -799 bp of the CCTalpha proximal 5'-flanking region. Exposure of cells to a P. aeruginosa mutant strain producing alginate reduced CCTalpha promoter activity, whereas these effects were not observed in strains defective in alginate synthesis. Murine type II cells isolated from P. aeruginosa-infected CCTalpha promoter-beta-galactosidase transgenic mice exhibited significantly reduced CCT and beta-galactosidase enzyme activities versus control. Thus, a mucoid P. aeruginosa strain reduces mRNA synthesis of a key biosynthetic enzyme thereby decreasing levels of surfactant.

Endogenous beta-galactosidase expression in murine pancreatic islets

To the Editor: The tissue-/cell-specific recombinase system, the Cre/loxP system, has become a very useful tool to conditionally inactivate or activate gene function [1–6]. This system is based on the ability of Cre to catalyse the excision of DNA flanked by loxP site recognition sequences. A number of studies on cell lineage in pancreatic cells have been performed by intercrossing cell-specific Cre-expressing transgenic mice and ROSAβgeo26 (ROSA26) mice, which carry the gene for bacterial beta-galactosidase (lacZ) [7]. In the double transgenic offspring, lacZ is expressed where Cre was expressed at a high enough level to excise the floxed stop codon. Commonly, X-gal staining is used to detect beta-galactosidase activity: beta-galactosidase catalyses the hydrolysis of X-gal to an indolyl alcohol, which then oxidises to form an intense blue indigo stain. While specific induction of lacZ by precise control of Cre, as well as leakage of beta-galactosidase by spontaneous Cre excision in double transgenic mouse, have been well studied, little work has dealt with the contribution of endogenous beta-galactosidase activity to false-positive results. Heart, liver, spleen, kidney, brain and skeletal muscle from mouse, rat and rabbit [8], and cultured human foetal pancreas [9], have been studied for endogenous betagalactosidase activity [8]. In cultured human foetal pancreas, the mRNA expression and enzyme activity of beta-galactosidase are developmentally regulated, peaking at 24 weeks of gestation and declining to low levels in the adult [9]. Endogenous beta-galactosidase activity in these organs was rarely detected following incubation at a pH greater than 7.5. In contrast, bacterial beta-galactosidase activity in the ROSAβgeo26 was strongly detected at pH 8.0–9.0 [8]. In the mouse pancreas, we evaluated endogenous beta-galactosidase expression and studied factors that may contribute to false-positive results. Firstly, to determine expression of the endogenous betagalactosidase mRNA in various tissues, we performed RTPCR on cDNAs from mouse islets, liver, kidney and spleen as well as from pancreatic beta cells from mouse insulin promoter I (MIP)–green fluorescent protein (GFP) mice [10], purified with FACS by their GFP expression (Fig. 1). The sizes of the PCR products for beta-galactosidase and the internal control TATA-binding protein were 330 and 190 bp, respectively. Pancreatic beta cells and islets as well as other organs expressed endogenous beta-galactosidase (Glb1) mRNA (Fig. 1). Since it is important to determine endogenous betagalactosidase enzyme activity, we performed X-gal staining on sections of pancreas and kidney from normal adult C57BL/6 mice; endogenous beta-galactosidase is known to be expressed in the kidney. From mice cardiac-perfused

Characterization of Tetracycline-Inducible BitransgenicKrt12rtTA/+/tet-O-LacZMice

To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline. A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow. Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet. A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.

Positive Selection System for Identification of Recombinants Using α-Complementation Plasmids

A number of selection systems have been developed for direct selection of recombinant plasmids in cloning experiments (positive selection). In this study, the Commonly used LacZ-based alpha-complementation plasmid vectors have been used for designing a positive selection system for the selection of recombinants. The basis for the Strategy is the phenomenon of galactose sensitivity exhibited by galactose epimerase (galE) mutants of Escherichia coli. It is known that lacZ+ galE, but not lacZ- galE cells are killed upon addition of lactose due to the accumulation of a toxic intermediate, UDP-galactose, by hydrolysis of lactose. Using a galE mutant strain of E. coli that carries the lacZAM15 allele, various alpha-complementation plasmids that vary in their copy number were examined for their ability to be killed following addition of lactose. The results show that some plasmids that exhibit relatively high beta-galactosidase enzyme activity can be used effectively for positive selection. This selection would be extremely useful during primary cloning experiments such as construction of genomic or cDNA libraries and also in instances involving selection for rare recombinants.

Selective cell dissemination into the heart by retrograde intracoronary infusion in the rat.

Cell transplantation through the intracoronary route has theoretical advantages over direct intramuscular injection in global cell dissemination into the myocardium with less injury. The authors have established a novel experimental model of retrograde intracoronary infusion in the rat by which the feasibility of this infusion for cell transplantation and the dynamics of myoblasts grafted by this route were investigated. After retrograde intracoronary implantation of beta-galactosidase-expressing myoblasts, myoblasts were observed to disseminate to all layers of only the left ventricular free wall, with little myocardial damage, and to differentiate into multinuclear myotubes by day 28. Beta-galactosidase enzyme activity estimated that 31.4% of initially grafted myoblasts existed within the heart at 10 min after implantation. The number slightly increased by day 3, increased to 117.3% at day 7, and reached a plateau of 132.1% at day 14. These data suggest the utility of retrograde intracoronary infusion for selective cell dissemination into the myocardium.

Gene transfer into human keloid tissue with adeno-associated virus vector.

BACKGROUND Gene transfer is a new territory for clinicians. Intractable disorders might be approached in such a way. Adeno-associated virus (AAV) vector has been transfected successfully into a variety of tissues including skin. We evaluated the ability of this vector to transfer and cause expression of the reporter gene in human keloid tissue. METHODS Human keloid specimens were injected with an AAV vector encoding beta-galactosidase and incubated for 4 weeks after injection. The presence of mRNA and beta-galactosidase enzymatic activity were assayed by reverse-transcriptase polymerase chain reaction and the X-gal technique. RESULTS Gene expression shown by reverse-transcriptase polymerase chain reaction was observed in keloid tissue 4 weeks after injection, and so was the positive X-gal staining. CONCLUSION Our results showed that AAV vector could transduce human keloid tissue effectively. Replacement of the reporter gene with a functioning gene might be feasible for keloid treatment.

A 3-kilobase region derived from the rat cathepsin L gene directs in vivo expression of a reporter gene in sertoli cells in a manner comparable to that of the endogenous gene.

During mammalian spermatogenesis, the transcription of several genes in Sertoli cells is turned on and off as the adjacent male germ cells progress through the stages of the cycle of the seminiferous epithelium. A requirement for defining how germ cells regulate this process is the identification of a promoter that confers, in vivo, accurate, stage-specific gene expression in Sertoli cells. To date, such a promoter has not been identified. Using transgenic mice, we show that the 3-kilobase genomic fragment immediately upstream of the rat cathepsin L translation start site directs expression of the reporter gene, beta-galactosidase, only in Sertoli cells. The expression pattern of the reporter gene recapitulated that of the endogenous gene in Sertoli cells as 75% of the seminiferous tubules that contained X-gal positive Sertoli cells were at stages VI-VIII and beta-galactosidase enzymatic activity was 4-fold higher in mature testes compared with immature testes. This is, to our knowledge, the first identification of a promoter region that contains all of the regulatory elements required for accurate, stage-specific gene expression in Sertoli cells.

Mutations in the N-terminus of VP5 alter its interaction with the scaffold proteins of herpes simplex virus type 1.

During the assembly process of herpes simplex virus type 1 capsids, there is an essential interaction between the C-terminal tail of the scaffold proteins (22a and 21) and the major capsid protein (VP5). Recent studies of spontaneous revertant viruses that overcome a blocked maturation cleavage site of the scaffold proteins have shown that the N-terminus of VP5 is important for this interaction. One of the revertant viruses, PR7, encodes a second-site mutation at residue 69 of VP5 which unlike wild-type VP5 fails to interact with 22a and thus gives white colonies in the yeast two-hybrid assay. In the present study a small DNA fragment, encoding residues 1 to 85 of wild-type and PR7 VP5, was mutagenized using error-prone PCR. Mutagenized DNA was used in the yeast two-hybrid assay to identify mutations in wild-type VP5 that resulted in loss of 22a binding (white colonies), or in PR7 VP5 that resulted in a gain of function (blue colonies). For the loss of function experiments, using KOS VP5, a row of eight thymidine nucleotides (codons 37-40) resulted in many frameshift mutations, which led us to terminate the study without reaching a statistically significant result. For the PR7 experiment, 30 clones were identified that had single amino acid substitutions, and these mutations were localized to amino acids 27-45 and 63-84 of VP5. The most frequent mutation was a reversion back to wild-type. The next most frequent were E28K and N63S, and these gave the highest beta-galactosidase enzyme activities (indicative of PR7VP5-22a interaction), 30 and 20% of wild-type, respectively. When E28K and N63S were transferred into the wild-type VP5 background, that is, in the absence of the PR7 mutation, they gave rise to different phenotypes. The E28K mutation lost its ability to interact with the scaffold proteins as judged by this assay. Therefore, it may be acting as a compensatory mutation whose phenotype is only expressed in the presence of the original PR7 mutation. However, the N63S mutation in the wild-type VP5 background increased the interaction, as judged by the beta-galactosidase activity, by a factor of 9 relative to when the PR7 mutation was present. Even more surprising, in the absence of the PR7 mutation the enzyme activity was still greater, by a factor of 2, than that observed for wild-type VP5. This study provides further evidence that the N-terminus of VP5 is in intimate association with the C-terminus of the scaffold proteins.

Conditional Gene Expression in the Epidermis of Transgenic Mice Using the Tetracycline-Regulated Transactivators tTA and rTA Linked to the Keratin 5 Promoter

To produce conditional expression of genes in the mouse epidermis we have generated transgenic mouse lines in which the tetracycline-regulated transcriptional transactivators, tTA and rTA, are linked to the bovine keratin 5 promoter. The transactivator lines were crossed with the tetOlacZ indicator line to test for transactivation in vivo. In the absence of doxycycline, the K5/tTA line induced beta-galactosidase enzyme activity in the epidermis at a level 500-fold higher than controls, and oral and topical doxycycline caused a dose- and time-dependent suppression of beta-galactosidase mRNA levels and enzyme activity. In the K5/rTA lines, doxycycline induced beta-galactosidase activity between 3- and 50-fold higher depending on the founder line, and this occurred within 24-48 h after dosing. Histochemical analysis of all lines localized beta-galactosidase expression to the basal layer of the epidermis and the outer root sheath of the hair follicle, as well as other keratin 5 positive tissues. In several K5/rTA lines, skin-specific transactivation was restricted to the hair follicle. Treatment of these double transgenic mice with 12-O-tetradecanoyl-phorbol-13-acetate caused rapid migration of beta-galactosidase marked cells from the hair follicle through the interfollicular epidermis, demonstrating the usefulness of this specific double transgenic for fate mapping cells in the epidermis. These results show that the tetracycline regulatory system produces effective conditional gene expression in the mouse epidermis, and suggest that it should be amenable to suppression and activation of foreign genes during development and specific pathologic conditions relevant to the epidermis.

Functional Analysis of the Heme Oxygenase‐1 Gene Promoter

Basal and inducer-dependent transcription activity of the heme oxygenase-1 gene promoter can be measured using the luciferase reporter gene in transfection assays. Methods are provided in this unit for purification of plasmid DNA, transient and stable transfection of DNA into cells, and measurement of luciferase and beta-galactosidase enzyme activities using chemiluminescent-based assays. These protocols can be used to identify inducer-activated cis elements.