Understanding the evolution and spread of chikungunya virus in the Americas using complete genome sequences.

Local transmission of chikungunya virus (CHIKV) was first detected in the Americas in December 2013, after which it spread rapidly throughout the Caribbean islands and American mainland, causing a major chikungunya fever epidemic. Previous phylogenetic analysis of CHIKV from a limited number of countries in the Americas suggests that an Asian genotype strain was responsible, except in Brazil where both Asian and East/Central/South African (ECSA) lineage strains were detected. In this study, we sequenced thirty-three complete CHIKV genomes from viruses isolated in 2014 from fourteen Caribbean islands, the Bahamas and two mainland countries in the Americas. Phylogenetic analyses confirmed that they all belonged to the Asian genotype and clustered together with other Caribbean and mainland sequences isolated during the American outbreak, forming an 'Asian/American' lineage defined by two amino acid substitutions, E2 V368A and 6K L20M, and divided into two well-supported clades. This lineage is estimated to be evolving at a mean rate of 5 × 10-4 substitutions per site per year (95% higher probability density, 2.9-7.9 × 10-4) and to have arisen from an ancestor introduced to the Caribbean (most likely from Oceania) in about March 2013, 9 months prior to the first report of CHIKV in the Americas. Estimation of evolutionary rates for individual gene regions and selection analyses indicate that (in contrast to the Indian Ocean Lineage that emerged from the ECSA genotype followed by adaptive evolution and with a significantly higher substitution rate) the evolutionary dynamics of the Asian/American lineage are very similar to the rest of the Asian genotype and natural selection does not appear to have played a major role in its emergence. However, several codon sites with evidence of positive selection were identified within the non-structural regions of Asian genotype sequences outside of the Asian/American lineage.

Risks and music - patterns among young women and men in Sweden.

Music and high levels of sound have not traditionally been associated with risk-taking behaviors. Loud music may intensify and bring more power and meaning to the musical experience, but it can at the same time be harmful to hearing. The present study aims to increase the knowledge about young women's and men's risk judgement and behaviour by investigating patterns in adolescent risk activities among 310 adolescents aged 15-20 (143 women; 167 men). The Australian instrument ARQ was used with additional questions on hearing risks and a factor analysis was conducted. The main results showed that the factor structure in the judgement and behavior scale for Swedish adolescents was rather different from the factor structure in the Australian sample. Also, the factor structure was not similar to the Australian sample split on gender. The results are discussed from a gender- and existential perspective on risk taking, and it is emphasized that research on risk behavior needs to reconceptualize stereotypical ideas about gender and the existential period in adolescence.

Hearing, use of hearing protection, and attitudes towards noise among young American adults.

The purpose of the present study was to investigate possible associations between college students' attitudes, risk-taking behaviour related to noisy activities, and hearing problems such as threshold shifts or self-experienced hearing symptoms. The sample included 258 students aged between 17 and 21 enrolled at the University of Pennsylvania, USA. A questionnaire measuring attitudes towards noise, use of hearing protection, and self-reported hearing symptoms was distributed among the students. After completing the questionnaire a hearing screening, including pure-tone audiometry and tympanometry, was conducted. The result revealed that 26% had thresholds poorer than the screening level of 20 dBHL. Attitudes were significantly related to self-experienced hearing symptoms, but not to threshold shifts. Attitudes and noise sensitivity was, significantly related to use of hearing protection. Hearing protection use was found in activities such as using firearms, mowing lawns, and when using noisy tools but was less reported for concerts and discotheques. It can be concluded that the young adults in this study expose themselves to hearing risks, since the use of hearing protection is in general very low.

Reported hearing protection use in young adults from Sweden and the USA: effects of attitude and gender.

The present study investigates differences between a Swedish and an American sample of young students regarding attitudes towards noise and the use of hearing protection at concerts. The study population was comprised of 179 participants from Sweden and 203 participants from the United States, who ranged in age from 17 to 21 years. Questionnaires were used to gather information on hearing symptoms and attitudes towards noise (Youth Attitude to Noise Scale). Multivariate analysis of variance revealed that attitudes towards noise differed significantly due to gender and country. Men had slightly more positive attitude towards noise than women, and men from the USA had more positive attitudes than men from Sweden. Least positive were the women from Sweden (except regarding attitudes towards the ability to concentrate in noisy environments). Multivariate logistic regression analysis was used to examine the influence of attitudes towards noise and country on young people's use of hearing protection at concerts. The results indicated that attitudes and country explained 50% of the variance in use of hearing protection.

The influence of socio-economic status on adolescent attitude to social noise and hearing protection.

The focus of the present study, of 1285 adolescents, was young people's attitudes towards noise and their use of hearing protection at discos and pop concerts. Comparisons were made between adolescents from different age groups, and with different socio-economic status. Logistic regressions indicated that "worry before attending noisy activities" and "hearing symptoms" such as tinnitus and noise sensitivity could, to some degree, explain the use of hearing protection in noisy environments. Another conclusion to be drawn from this study was that adolescents' attitudes and behaviours regarding hearing protection use differed between levels of socio-economic status. Individuals with high SES expressed more negative attitudes and used ear protection to a greater extent than those with lower SES. This result might indicate differences in the development of future auditory problems among individuals with different levels of socio-economic status. The cause of hearing impairment and tinnitus may not be restricted merely to noise exposure. Psychological aspects, such as attitudes towards noisy environments and the individual's behaviour regarding the use of hearing protection may be considered as important factors in the understanding of why the prevalence of hearing related problems has increased among adolescents.

Self-reported tinnitus and noise sensitivity among adolescents in Sweden.

It seems to be a common opinion among researchers within the field of audiology that the prevalence of tinnitus will increase as a consequence of environmental factors, for example exposure to loud noise. Young people are exposed to loud sounds, more than any other age group, especially during leisure time activities, i.e. at pop concerts, discotheques and gyms. A crucial factor for the prevention of hearing impairments and hearing-related symptoms in the young population is the use of hearing protection. The focus of the present study is use of hearing protection and self-reported hearing-related symptoms, such as tinnitus and noise sensitivity in a young population of high-school students (N=1285), aged 13 to 19 years. The results show that the prevalence of permanent tinnitus and noise sensitivity, reported in the total group, was 8.7% and 17.1% respectively. Permanent tinnitus was not significantly related to level of socio-economic status, but age-related differences in the prevalence rates of experienced tinnitus and noise sensitivity were found to be significant. Older students reported such symptoms to a greater extent than younger students did. Those who reported tinnitus and other hearing-related symptoms protected their hearing to the highest extent and were the ones most worried.

Molecular cloning and high-level expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes.

The cDNA encoding the human polymerase beta from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase beta cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase beta mRNA in Escherichia coli and all yielded a high level of human polymerase beta. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.

Cloning of the functional promoter for human insulin-like growth factor binding protein-4 gene: endogenous regulation.

The majority of the colon cancers analyzed to-date express insulin-like growth factor binding protein (IGFBP)-4, and antisense inhibition of IGFBP-4 messenger RNA (mRNA) confers a growth advantage to the cells in response to endogenous and exogenous IGFs. We recently reported a significant up-regulation of IGFBP-4 expression in a human colon cancer cell line (CaCo2) on spontaneous differentiation of the cells in culture. This suggests that the expression of IGFBP-4 may be related to growth and differentiation of colon cancer cells. To study the endogenous factors involved in the transcriptional regulation of IGFBP-4, we have isolated and sequenced the human (h) IGFBP-4 promoter. The approximately 1.3 kilobase pair (kb) 5' flanking region of the IGFBP-4 gene is GC rich and possesses several potential regulatory elements. These elements include a typical TATA box with sequence TATAA, located -299 nt from the initiation ATG codon. The cap site is located 14 nt downstream of the TATA box as determined by primer extension analysis. A 1.4-kb DNA fragment including the 1.254 kb 5' flanking region of the hIGFBP-4 gene was subcloned into a luciferase reporter vector (pGL-2 basic) either in the sense (BP-4-S-pGL) (S) or antisense (BP-4-AS-pGL) (AS) (negative control) orientation, relative to the luciferase coding sequence in the vector. CaCo2 cells were transfected with either the S or the AS vectors on days 2-10 of culture; cotransfection with the SV40-beta-Galactidose (Gal) vector was used to correct for transfection efficiency. The ratio of luciferase/beta-Gal expression by CaCo2 cells transfected with the S vectors increased significantly from days 3 and 4 to days 5 and 6 of culture, followed by a sharp decline on days 7-9, resembling the pattern of endogenous expression of IGFBP-4 by the cells; the expression of luciferase by the AS vectors remained low and insignificant. These results thus suggest that the approximately 1.4 kb 5' flanking region of the IGFBP-4 gene contains the cis elements required for regulation of the IGFBP-4 gene. Cloning and sequencing of the functional hIGFBP-4 promoter will enable us, for the first time, to study the endogenous factors/mechanisms responsible for the growth/differentiation (cell density) associated regulation of IGFBP-4 expression in colonic epithelial cells.

Episomal expression of sense and antisense insulin-like growth factor (IGF)-binding protein-4 complementary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I.

HT-29 cells express and secrete insulin-like growth factor (IGF)-II and only one of the six IGF-binding proteins, IGFBP-4. In the present study, the physiological role of endogenous IGFBP-4 in regulating the growth response of HT-29 cells to exogenous and endogenous IGFs was examined. Both the basal and the IGF-stimulated growth of HT-29 cells was significantly increased over control values in the presence of IGFBP-4 antibody, suggesting that endogenous IGFBP-4 is a potent inhibitor of the mitogenic effects of endogenous and exogenous IGFs. In order to further confirm the inhibitory role of endogenous IGFBP-4, sense and antisense complementary DNA fragments of human IGFBP-4 were ligated into an episomal mammalian expression vector (pCEP4). Restriction mapping and Southern blot analysis were used to confirm directional cloning of the IGFBP-4 complementary DNA fragments in the sense and antisense directions in the pCEP4 vectors. HT-29 cells were transfected with either the control (no insert, C-P), sense (S-P), or antisense (AS-P) vectors and subjected to hygromycin selection. The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in the conditioned media (CM) of 10(7) cells by ligand and immunoblot analysis. IGFBP-4 concentrations were 7.4 +/- 1.7-fold higher in the CM of S-P cells compared to that in the CM of C-P cells, while IGFBP-4 concentrations in the CM of AS-P cells were significantly lower than those present in the CM of C-P cells. Both the basal and the IGF-I-stimulated growth of the AS-P cells was significantly higher than that of the C-P and S-P cells. The basal (non-stimulated) and the IGF-I-stimulated growth of the S-P cells was not significantly different from that of the C-P cells, suggesting that overexpression of IGFBP-4 was not inhibitory to the growth of the HT-29 cells. The basal growth of the S-P and C-P cells was significantly increased in the presence of IGFBP-4 antibody, once again suggesting that endogenous IGFBP-4 was a potent inhibitor of autocrine effects of endogenous factors (IGF-II). Addition of IGFBP-4 antibody had no significant effect on the basal growth of the AS-P cells, confirming that the difference between the growth response of the AS-P, C-P, and S-P cells was largely contributed by the difference in the endogenous secretion of IGFBP-4 by the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The human DNA polymerase beta gene structure. Evidence of alternative splicing in gene expression.

DNA polymerase beta (beta-pol) is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. Using two human genomic libraries we have cloned the complete human beta-pol gene and determined the organization of the beta-pol coding sequence within the gene. The human beta-pol gene spans 33 kb and contains 14 exons that range from 50 to 233 bp. The 13 introns vary from 96 bp to 6.5 kb. Information derived from this study was used in defining the location of a deletion/insertion type restriction fragment length polymorphism (RFLP) 5' to exon I of the human beta-pol gene. This RFLP was utilized in linkage analysis of DNAs from CEPH families and the results confirm the previous assignment of the human beta-pol gene to chromosome 8 (p12-p11). Analysis of mRNA from six human cell lines using the polymerase chain reaction showed the expression of two beta-pol transcripts. Sequence analysis revealed that the size difference in these transcripts was due to deletion of the 58 bp sequence encoded by exon II, suggesting that the smaller transcript results from an alternative splicing of the exon II sequence during processing of the beta-pol precursor RNA.

RNA polymerase II transcription. Rate of promoter clearance is enhanced by a purified activating transcription factor/cAMP response element-binding protein.

The core promoter of the human DNA beta-polymerase (beta-pol) gene is regulated by proteins binding at 3 GC boxes and the single activating transcription factor/cAMP response element (ATF/CRE) centered at -45; the central 8 residues of this ATF/CRE match the ATF/CRE consensus sequence, TGACGTCA. Previously, we purified a beta-pol promoter ATF/CRE-binding protein (named palindrome-binding protein or PBP) from bovine testes and found that this protein is a beta-pol promoter transcriptional activator in vitro using a HeLa nuclear extract transcription system (Widen, S. G., and Wilson, S. H. (1991) Biochemistry 30, 6296-6305). In this study, we determined the mechanism of in vitro transcriptional activation by this purified PBP. We used a PBP-depleted HeLa nuclear extract transcription system with an artificial promoter containing a solitary activator element corresponding to the entire 22-nucleotide beta-pol promoter ATF/CRE-binding site. Kinetic analyses of the 180-nucleotide run-off product formation indicated that stimulation of transcriptional activity by PBP was due entirely to an increase in the rate constant for promoter clearance. Thus, under our conditions, the purified PBP had no effect on the rate of closed preinitiation complex formation or for the closed complex to open complex transition. Instead, the rate of productive initiation leading to the 180-nucleotide transcript was stimulated by PBP. We found that the rate of closed preinitiation complex formation was not in rapid equilibrium with promoter and RNA polymerase II, in contrast to the model with prokaryotic RNA polymerase transcription. The results also indicated that PBP binding to the ATF/CRE is required for the stimulation of promoter clearance. These studies define the kinetic mechanism of a purified ATF/CRE-binding protein in stimulation of the in vitro transcription of a designed mammalian promoter.

Expression and purification of biologically active domain I of the human polymeric immunoglobulin receptor.

Previous studies using proteolytic fragments and synthetic peptides have indicated that domain I of human polymeric immunoglobulin receptor (PIgR) is necessary for ligand binding. The expression in E. coli, and subsequent IgM-affinity purification of domain I of human PIgR is described. The recombinant domain I protein (rDI) was similar in structure to native SC domain I in that it bound specifically to MAb 6G11, an antibody which recognizes a critical portion of the PIg binding site in domain I. The biological activity of rDI was indicated by high affinity binding to PIgA (Kd = 1.6 x 10(-7) M) and IgM (Kd = 5.1 x 10(-7) M). Domain I of human SC is therefore sufficient for binding to PIg.

Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme.

We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.

Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase beta by UV cross-linking.

Rat DNA polymerase beta (beta-pol) is a 39-kDa monomeric protein, organized in two structurally and functionally distinct domains. The 8-kDa NH2-terminal domain binds single-stranded (ss) DNA, whereas the 31-kDa COOH-terminal domain does not. To facilitate studies on ssDNA binding structure-function relationships of beta-pol, we overexpressed the 8-kDa domain in Escherichia coli, and purified the recombinant protein to homogeneity. Single-stranded nucleic acid binding of the recombinant 8-kDa domain was found to be similar to that previously reported for the 8-kDa fragment prepared by proteolysis of intact beta-pol (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P. Karpel, R. L., and Wilson, S. H. (1990b) J. Biol. Chem. 265, 2124-2131; Casas-Finet, J. R., Kumar, A., Morris, G., Wilson, S. H., and Karpel, R. L. (1991) J. Biol. Chem. 266, 19618-19625). Residues in or near the DNA-binding pocket of the recombinant 8-kDa domain were examined by photochemical cross-linking to [32P] p(dT)16. Cross-linking was localized to a tryptic fragment spanning residues 28 through 35 and a V8 protease fragment spanning residues 27 through 58. Sequence analysis of the various [32P]p(dT)16-labeled proteins indicated that Ser30 and His34 were modified by cross-linking to p(dT)16. Therefore, these residues of the ssDNA-binding domain of beta-pol appear to be in close contact with this nucleic acid probe.

Identification of a nuclear protein binding element within the rat brain protein kinase C gamma promoter that is related to the developmental control of this gene.

Protein kinase C gamma (PKC gamma) is a brain-specific isozyme expressed at a high level in the adult but not in the fetal or newborn rat. At least seventeen nuclear protein binding sites within the 5'-flanking region extending from -1612 to +243 had been identified by DNase I footprinting analysis and gel mobility shift assays. Among them, one site, GAATTAATAGG, at -669 to -679 is protected from DNase I digestion by nuclear protein from newborn but not from the adult rat brain. The levels of this binding protein, as determined by gel mobility shift assay, were found inversely related to the levels of PKC gamma in rat brain at different stages of development. These results suggest that this particular binding site may participate in the developmental regulation of PKC gamma gene.

Protein-protein interactions of HIV-1 reverse transcriptase: implication of central and C-terminal regions in subunit binding.

Human immunodeficiency virus 1 reverse transcriptase (RT) purified from virions is composed of a approximately 51,000 Mr polypeptide and a approximately 66,000 Mr polypeptide that are thought to be in heterodimer structure (Chandra et al., 1986; Hansen et al., 1988; Starnes & Cheng, 1989) and are identical except for a 15,000 Mr C-terminal truncation in the smaller species (Di Marzo-Veronese et al., 1986). We prepared individual bacterial-recombinant RTs as the approximately 66,000 Mr polypeptide (p66) or as the approximately 51,000 Mr polypeptide (p51) and then conducted various in vitro protein-protein binding experiments. Analytical ultracentrifugation studies in 0.25 M NaCl at pH 6.5 revealed that p66 was in monomer-dimer equilibrium with KA of 5.1 x 10(4) M-1. p51 failed to dimerize and behaved as a monomer under these conditions. Mixing of the p66 and p51 polypeptides resulted in a 1:1 heterodimer with KA of 4.9 x 10(5) M-1. These results on formation of the p66/p66 homodimer and p66/p51 heterodimer were confirmed by gel filtration analysis using FPLC Superose-12 columns. Binding between p66 and individual p66 segment polypeptides also was observed using an immunoprecipitation assay. Binding between p51 and p66 in this assay was resistant to the presence of approximately 1 M NaCl, suggesting that the binding free energy has a large hydrophobic component. C-Terminal truncation of p66 to yield a 29-kDa polypeptide eliminated binding to p66, and N-terminal truncation of p66 to yield a 15-kDa peptide also eliminated binding to p66. The results indicate that purified individual RT peptides p51 and p66 are capable of binding to form a 1:1 heterodimer and suggest that the central region of p66 is required for this subunit binding; the C-terminal region (15,000 Mr) of p66 appears to be required also, as p51 alone did not dimerize.

Mammalian beta-polymerase promoter: large-scale purification and properties of ATF/CREB palindrome binding protein from bovine testes.

The mammalian DNA repair enzyme beta-polymerase is encoded by a single-copy gene that is expressed in all tissues and cell lines studied to date. A protein fraction with high binding affinity for an ATF/CREB-like binding element, GTGACGTCAC, at -49 to -40 in the core beta-polymerase promoter has been purified to near-homogeneity from a nuclear extract of bovine testes. The major binding activity, as monitored by gel mobility shift assay, is recovered in 20% yield by a procedure involving oligonucleotide affinity chromatography. The purified protein yields DNase I footprinting and gel shift binding patterns indistinguishable from the activity in crude extracts. The final fraction activates transcription in an in vitro transcription reaction. The native molecular weight of the purified binding activity is about 100-120K as measured by gel filtration. SDS-PAGE of the purified fraction revealed that it contains several polypeptides in the molecular weight range of 30-52K, yet two of these peptides (Mr 49K and 52K) are predominant. Specific binding to the palindrome is salt-sensitive and is consistent with the formation of nine ion pairs (from log KA vs log KCl plots) and has a KA at 200 mM KCl of 5.8 X 10(11) M-1. Kinetic studies with synthetic oligonucleotides as binding ligands indicate that the purified protein can bind tighter to or discriminate between the beta-polymerase ATF/CREB element and similar elements derived from somatostatin and chorionic gonadotropin genes.

Mammalian beta-polymerase promoter: phosphorylation of ATF/CRE-binding protein and regulation of DNA binding.

The gene for the mammalian DNA repair enzyme DNA polymerase beta (beta-pol) is constitutively expressed in most cells, but is regulated in a tissue-specific fashion and can be induced in response to some types of DNA damaging agents. The promoter for the human beta-pol gene has been characterized and found to be TATA-less, but it does have multiple GC boxes and one ATF/CRE-binding site located within 50 residues 5' of the major mRNA start site. The ATF/CRE-binding site has been found to be essential for activity of the cloned promoter. We report that a bovine testes DNA-binding protein with specificity for the beta-pol promoter ATF/CRE-binding site is phosphorylated in vivo and contains several phosphorylation sites. Sequence specific DNA-binding by the purified protein is reduced when the natural protein is dephosphorylated or when it is hyperphosphorylated by protein kinase A (cKA) in vitro. These results suggest the possibility that phosphorylation systems may change binding of this ATF/CRE-binding protein to the beta-pol promoter and in turn modulate the promoter. Possible correlation of the results with transient expression activity of the cloned beta-pol promoter fusion gene was obtained in 293 cells. Cotransfection with a cKA expression plasmid to elevate phosphorylation was found to strongly reduce promoter activity.

The ATF/CREB transcription factor-binding site in the polymerase beta promoter mediates the positive effect of N-methyl-N'-nitro-N-nitrosoguanidine on transcription.

DNA polymerase beta (pol beta) is a constitutively expressed DNA repair enzyme in vertebrate cells. Yet, it had been shown previously that the pol beta mRNA level increases in Chinese hamster ovary (CHO) cells within 4 h after treatment with several monofunctional DNA damaging agents, notably, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Herein we report that a transfected pol beta promoter fusion gene is activated by MNNG treatment of CHO cells; mRNA from the transfected gene is approximately 10-fold higher in treated cells than in untreated cells 16 h after treatment. This activation is mediated through the decanucleotide palindromic element GTGACGTCAC at positions -49 to -40 in the "TATA-less" core promoter. This element, which is similar to the ATF/CREB transcription factor-binding site in a number of mammalian genes, forms the center of a strong protein-binding site for CHO cell nuclear extract proteins. Mutated pol beta promoter fusion genes lacking the element fail to bind protein at this site and fail to respond to MNNG treatment of cells.

Characterization of the 5'-flanking region of the rat protein kinase C gamma gene.

The 5'-flanking region of protein kinase C (PKC) gamma gene was identified from a rat liver genomic library in a bacteriophage lambda Charon 4A. A 3.6-kilobase (kb) genomic fragment containing the 5'-flanking region, first exon, and first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 mapping and primer extension, was located 243 base pairs upstream from the translational initiation site. Promoter activity of a DNA segment spanning the 5'-flanking region was demonstrated by both in vitro transcription using HeLa cell nuclear extracts and chloramphenicol acetyltransferase assay by transfection of 293 cells with a PKC gamma-CAT fusion construct. Chloramphenicol acetyltransferase assay revealed that a fragment of about 0.16 kb from the transcriptional initiation site was sufficient for promoter activity in these cells, and the construct containing up to 1.6 kb from the cap site was expressed at a similar level. This promoter-active fragment contains several regions similar to defined transcriptional elements in other mammalian promoters, such as those for stimulatory protein 1 (Sp1), activator proteins 1 and 2 (AP1, AP2), c-myc, cAMP regulatory element-binding protein (CREB), and enhancer core (EnhC). Investigation of the genomic structure of PKC gamma gene may lead to the identification of cis-elements controlling tissue-specific and developmental stage-specific expression of PKC gamma.