Major Regulatory Region Research Articles

A role of transcription factors in pathogenic processes associated with schizophrenia

Schizophrenia is a severe mental illness, the etiology and pathogenesis of which are significantly contributed by hereditary factors. Genome-wide association analysis shows that the majority of genetic variants associated with a high risk of schizophrenia are located in regulatory regions of genes. In this brief review, data on the overall structure of the major regulatory regions of genes are summarized. The main focus is on enhancers, which are regions of the genome that enhance gene activity. The participation of transcription factors interacting with gene regulatory regions in various molecular and cellular processes, the disruption of which is associated with the pathogenesis of schizophrenia, is discussed in detail.

R-Loops and Mitochondrial DNA Metabolism.

R-loops forming inadvertently during transcription can threaten genome stability, but R-loops are also formed intentionally, as a means of regulating transcription and other aspects of DNA metabolism. The study of R-loops in mitochondria is in its infancy, and yet there is already clear evidence that they are predominantly located in the major regulatory region of the mammalianmitochondrial genome. Here, we describe how mitochondrial R-loops have been characterized to date, with the emphasis on the problems of their being extremely labile, and how to minimize their loss during extraction. The oft-overlooked issues of RNA-DNA hybrids not being synonymous with R-loops, and adventitious RNA hybridization to DNA, are tackled head on; and possible new approaches are described and placed in the context of future research lines that could reveal the detailed roles of R-loops in the metabolism of mitochondrial DNA.

Molecular basis of weak A subgroups in the Korean population: Identification of three novel subgroup-causing variants in the ABO regulatory regions.

Recent studies on Chinese and Japanese populations have shown that weak ABO subgroups could be caused by variants in the major regulatory regions of ABO, the proximal promoter, +5.8-kb site, and CCAAT-binding factor/NF-Y binding site. We investigated the molecular basis of weak A subgroups in the Korean population. This study included 11 samples suspected to have a weak A subgroup. These samples were subjected to sequencing analysis of ABO exons 6 and 7. If no subgroup-causing variants were detected in this region, exons 1-5 and three major regulatory regions were sequenced. Sequencing analysis of exons 6 and 7 detected two known subgroup alleles (ABO*AW.10, n=5; ABO*AEL.02, n=2). The remaining four samples contained a sequence variant in the proximal promoter (g.4944C>T, n=1; g.4954G>T, n=1) or +5.8-kb site (g.10843T>C, n=1; g.10935C>T, n=1). Notably, three of the four variants (g.4944C>T, g.4954G>T, and g.10843T>C) have not been reported previously in weak ABO subgroups. This study provides the first evidence that alterations in the proximal promoter and + 5.8-kb site could account for a substantial proportion of weak A subgroups in the Korean population.

Sequence variants in the proximal promoter and +5.8-kb site of ABO in Koreans with weak B phenotypes.

Several studies on Chinese and Japanese populations have revealed that a substantial proportion of weak B subgroups are caused by variants in the major regulatory regions of ABO, the proximal promoter, CCAAT-binding factor/NF-Y binding site and +5.8-kb site. We performed molecular analyses of these regions in Koreans with weak B phenotypes. This study included 16 samples with weak B phenotypes (4 B3 , 1 Bw , 5 A1 B3 and 6 A1 Bw ) harbouring no subgroup-causing variants in ABO exons 6 and 7. These samples were subjected to sequencing analysis of exons 1-5 and the major regulatory regions of ABO. Of the 16 samples, 14 were found to carry a sequence variant either in the proximal promoter (g.4991_5008del [n= 3]) or the +5.8-kb site (g.10893G>A [n= 4] and g.10925C>T [n= 7]). The remaining two samples were found to contain no subgroup-causing variants. Our study demonstrates that sequence variants in the proximal promoter and +5.8-kb site account for a substantial proportion of weak B subgroups in Koreans, suggesting that molecular analysis of these regions is essential for the accurate determination of ABO genotypes in Koreans with weak B phenotypes.

Laboratory-Developed Tests: Design of a Regulatory Strategy in Compliance with the International State-of-the-Art and the Regulation (EU) 2017/746 (EU IVDR [In Vitro Diagnostic Medical Device Regulation

PurposeThis study aimed at the development of a regulatory strategy for compliance of laboratory-developed tests (LDTs) with requirements of the Regulation (EU) 2017/746 (“EU-IVDR”) under consideration of international requirements for LDTs as established in major regulatory regions. Furthermore, it was analysed in how far elements of current LDT regulation could qualify for an internationally harmonised concept ensuring quality, safety and performance of LDTs.MethodsA review of regulatory literature including legislation as well as guidance documents was performed. The regulatory strategy was adapted from international guidance concepts used for commercially marketed IVD. It was then applied to the example of a large medical laboratory in the EU. A high-level comparison was conducted to identify gaps and matches between the different international regulatory requirements for LDTs.ResultsA four-step strategy for compliance of LDTs with the EU IVDR was implemented in an exemplary medical laboratory. On the basis of an internationally used LDT definition, LDTs constitute nearly 50% of the total IVD devices used in the laboratory. While an ISO 15189-compliant QMS is a major component, it should be accompanied by the application of appropriate processes for risk management, performance evaluation and continuous monitoring of LDTs. At least six criteria represent common characteristics of a potential, internationally convergent concept for the regulation/standardization of LDTs.ConclusionsThis study confirms the impact of LDTs for individualized and innovative medical laboratory testing. Prerequisites for LDT use as especially given by the IVDR and missing interpretation in the EU with regard to the scope of LDT definition, the application of standards and the extent of documentation for LDTs currently lead to uncertainties for both laboratories and regulatory bodies responsible for LDT oversight. The characteristics identified as common criteria for ensuring quality, safety and performance of LDTs may be considered as central elements of future international consensus guidance.

P01.27 Repressive histone marks (H3K27me3) and DNA hypermethylation suggests epigenetic regulation of miR-379/miR-410 (C14MC) cluster in oligodendrogliomas

Abstract MicroRNAs(miRNAs) are often grouped into clusters in the genome and dysregulation of miRNA clusters has been shown in various cancers. miR-379/miR-656 cluster(C14MC) is one of the largest miRNA clusters located within an imprinted chromosomal region, Dlk1-Dio3 locus. Downregulation of miRNAs from C14MC has been observed in several CNS tumors, including GBM. However, status of expression of entire C14MC miRNA cluster and its clinicopathological significance in Oligodendrogliomas(ODGs) remains unexplored. Expression profile of C14MC miRNAs was performed on 44 ODGs using TaqMan low-density array (TLDA). Majority of C14MC miRNAs were observed to be significantly down-regulated in ODGs which was further validated on larger sample size using TCGA datasets. 17 miRNAs of C14MC were significantly dysregulated between grade II and III of ODGs in TLDA data, however TCGA dataset did not show any dysregulated miRNAs between the grades in ODGs. MEF2 has been reported as key transcription regulator for C14MC in rat neurons and muscle cell. Hence ChIP was performed for MEF2 binding in HS683 ODG cell line and we observed binding of MEF2A upstream of C14MC. Down-regulation of MEF2A was noted in both in-house and REMBRANDT dataset in ODGs. As per the TCGA methylation analysis of genomic region encompassing C14MC, we noted hypermethylation of this region in ODG. Treatment with the demethylating drug 5-AZA-cytidine resulted in re-expression of several miRNAs from C14MC in HS683 cells, thus suggesting the role of DNA methylation in regulation of C14MC. Further MEG3, a long noncoding gene located upstream of this cluster, showed downregulation in ODG (both in-house data and REMBRANDT) and also correlated with decrease in C14MC miRNA expression. Treatment of HS683 cell line with 5-aza-dC did not significantly induce MEG3 expression, further supporting contention that MEG3 region acquires a repressive chromatin state in ODGs. ChIP analyses revealed the predominance of H3K27me3 at the MEG3 DMR in the HS683 cell line suggesting a strong shift towards repressive histone modifications across major regulatory regions in MEG3. This further suggests involvement of epigenetic regulation of MEG3 expression in ODGs. Survival analysis of TCGA dataset demonstrated 59% prognostic correlation with C14MC miRNAs in ODGs. To conclude, this is the first report demonstrating the silencing of the second largest microRNA cluster in ODGs, implicating its tumor-suppressive role in their pathogenesis. Dysregulation of the transcription factor, particularly MEF2, along with epigenetic machinery involving DNA methylation and repressive histone modifications H3K27 mediated MEG3 downregulation could be the possible mechanism for C14MC downregulation, acting independently or in synergy. Further prognostic significance of this cluster is also highlighted.

In vivo methylation of mtDNA reveals the dynamics of protein–mtDNA interactions

To characterize the organization of mtDNA–protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in transcription and replication. The transcription termination region (TERM) within the tRNALeu(UUR) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL). Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that nucleoids are dynamically associated with proteins.

Vascular-brain signaling in hypertension: Role of angiotensin II and nitric oxide

Paracrine signaling by nitric oxide (NO) released from microvasculature within the brain affects multiple neuronal functions. Reviewed here is a role in central cardiovascular control. Within the nucleus tractus solitarii (NTS), a major regulatory region for arterial pressure, angiotensin II stimulates NO generation from endothelial nitric oxide synthase (eNOS). This enhances c-aminobutyric acid release to depress baroreflex function. In the spontaneously hypertensive rat (SHR), eNOS mRNA in the NTS is elevated compared to normotensive rats. Chronic inhibition of eNOS activity in the NTS of SHR reduced arterial pressure and increased baroreflex gain. Thus, eNOS-generated NO in the NTS plays a major role in control of baroreflex gain and arterial pressure. Indeed, its activity contributes to hypertension in the SHR. We propose that eNOS-generated NO in the SHR may be a compensatory mechanism for any potential threat to an adequate blood supply to the brain (eg, from genetically small arteries supplying the brainstem).

Alternative Promoter Use and Splice Variation in the Human Histamine H1 Receptor Gene

Upstream gene structure and mRNA expression of the human histamine H1 receptor gene was investigated in cells relevant to the pathogenesis of asthma, (primary cultured human airway smooth muscle (HASM) cells, primary cultured human bronchial epithelial cells and bronchial epithelial cell line [BEAS2B]), and other tissues known to express histamine H1 receptors (placenta and brain). Splice variation of the 5' terminal exons gave three separate locations for novel promoters upstream of the detected transcription start sites. Further splice variants in the 5' untranslated region were also observed. Transient transfections of promoter/luciferase constructs showed these regions directed expression in HASM cells and BEAS2B cells. Polymorphism screening of the major regulatory regions identified a number of novel single-nucleotide polymorphisms. Expression of splice variants was confirmed by real-time PCR assays. Results showed one 5' terminal exon splice variant, comprising exons B/K, expressed preferentially in all tissues. Interestingly, the other 5' terminal exon splice variants showed tissue-specific patterns of expression, with variant F/K expressed negligibly (0.1%) in HASM cells, but accounting for 19.3% and 8.3% of total expression in BEAS2B cells and differentiated human bronchial epithelial cells, respectively. Splice variant A/K was second most highly expressed in differentiated human bronchial epithelial cells (23%), whereas its expression in BEAS2B and HASM cells was 1.7% and 4.4%, respectively. These data suggest the use of alternative promoters directing human H1 receptor gene expression, both within and between cell types.

The proximal promoter of the human cathepsin G gene conferring myeloid-specific expression includes C/EBP, c-myb and PU.1 binding sites

Cathepsin G is a hematopoietic serine protease stored in the azurophil granules of neutrophil granulocytes. The mRNA of cathepsin G is transiently expressed during the promyelocyte stage of neutrophil maturation. The protease plays several roles in inflammatory actions of neutrophils, such as bactericidal effects. A human cathepsin G gene fragment of 6 kb directs a promyelocyte-specific expression in transgenic mice, indicating the presence of necessary cis-acting elements. However, neither the precise architecture of the promoter, nor the trans-acting factors responsible for its activation, have been characterized. In the present work, 2.6 kb upstream of the translation start site of the human cathepsin G gene was cloned. When transfected to monoblast-like U937 or to acute promyelocytic leukemia NB4 cells, both expressing endogenous cathepsin G, the initial 360 bp upstream of the translation start were sufficient to direct a strong expression of a luciferase reporter gene. No expression was observed in erythroid K562 control cells. Further deletions revealed three major regulatory regions containing the consensus binding-sites for the transcription factors C/EBP, c-myb and PU.1. Moreover, a GC-rich region, similar to a cis-element in the proteinase 3 promoter, was identified. Direct binding of the trans-factors C/EBPα, C/EBPɛ, c-myb and PU.1 to the promoter was shown by chromatin immunoprecipitation. The functional significance of the cis-elements was verified by site-directed mutagenesis. Mutations of the putative PU.1 site moderately decreased the activity of the promoter in monoblastic U937 cells, but not in promyelocytic NB4 cells. Separate mutations of the putative C/EBP binding site, c-myb-binding site or the GC-rich element resulted in a dramatically reduced transcriptional activity in both cell lines, suggesting cooperation between corresponding trans-factors.

Protein Kinase CK1δ Phosphorylates Key Sites in the Acidic Domain of Murine Double-Minute Clone 2 Protein (MDM2) That Regulate p53 Turnover

Murine double-minute clone 2 protein (MDM2) is an E3 ubiquitin ligase that regulates the turnover of several cellular factors including the p53 tumor suppressor protein. As part of the mechanism of p53 induction in response to DNA damage, a cluster of serine residues within the central acidic domain of MDM2 become hypophosphorylated, leading to attenuation of MDM2-mediated p53 destruction. In the present study, we identify the protein kinase CK1delta as a major cellular activity that phosphorylates MDM2. Amino acid substitution, coupled with phosphopeptide analyses, indicates that several serine residues in the acidic domain, including Ser-240, Ser-242, and Ser-246, as well as Ser-383 in the C-terminal region, are phosphorylated by CK1delta in vitro. We also show, through expression of a dominant negative mutant of CK1delta or treatment of cells with IC261, a CK1delta-selective inhibitor, that MDM2 is phosphorylated by CK1delta in cultured cells. These data establish the identity of a key signaling molecule that promotes the phosphorylation of a major regulatory region in MDM2 under normal growth conditions.

Methylation in the Core-promoter Region of the Chondromodulin-I Gene Determines the Cell-specific Expression by Regulating the Binding of Transcriptional Activator Sp3

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

Regulation of the cell expansion gene RHD3 during Arabidopsis development.

The RHD3 (ROOT HAIR DEFECTIVE3) gene encodes a putative GTP-binding protein required for appropriate cell enlargement in Arabidopsis. To obtain insight into the mechanisms of RHD3 regulation, we conducted a molecular genetic dissection of RHD3 gene expression and function. Gene fusion and complementation studies show that the RHD3 gene is highly expressed throughout Arabidopsis development and is controlled by two major regulatory regions. One regulatory region is located between -1,500 and -600 bp upstream of the RHD3 gene and is required for vascular tissue expression. The other region is intragenically located and includes the 558-bp first intron, which is responsible for high-level expression of RHD3 throughout the plant. The presence and location of this intron is essential for gene function because constructs lacking this intron or constructs with the intron in an abnormal position are unable to functionally complement the rhd3 mutations. We also analyzed the role of other RHD genes and the plant hormones auxin and ethylene in RHD3 regulation, and we determined that these act downstream or independently from the RHD3 pathway. This study shows that multiple levels of regulation are employed to ensure the appropriate expression of RHD3 throughout Arabidopsis development.

COUP-TF plays a dual role in the regulation of the ovalbumin gene.

The ovalbumin (Ov) gene contains a number of regulatory elements that control its transcriptional activity and restrict expression to avian oviduct. One major regulatory region, the steroid-dependent regulatory element (SDRE), is required for induction by estrogen and corticosterone. Another region, the negative regulatory element (NRE), downstream of the SDRE, acts primarily to repress gene expression. In addition, experiments within indicate that the binding site for the COUP transcription factor (COUP-TF) is also required for Ov gene transcription. To examine the interactions involving the SDRE, the NRE, and the COUP binding sites on Ov gene transcription, mutations in these regions were made and transfected into primary oviduct cell cultures. These experiments show that without the NRE, the SDRE is sufficient for induction by estrogen and corticosterone, irrespective of the COUP site. However, with the NRE intact, the COUP site is required for steroid induction, although without the NRE, the COUP site attenuates transcriptional activity. More interestingly, overexpression of COUP-TF1 with the Ov wild-type reporter construct alleviates the requirement for steroid hormones. These results demonstrate that the COUP site is essential and has a dual role in Ov gene transcription and that steroid hormones might directly or indirectly regulate the activity of COUP-TF1.

Role of the Sp Family of Transcription Factors in the Ontogeny of Growth Hormone Receptor Gene Expression

The growth hormone (GH) receptor is essential for the actions of growth hormone on postnatal growth and metabolism. GH receptor transcripts are characterized by the presence of disparate 5'-untranslated exons. Factors regulating the expression of the GC rich L2 transcript of the murine GH receptor gene have hitherto remained unidentified. To characterize the mechanisms regulating expression of the L2 transcript, primer extension and ribonuclease protection assays were used to identify transcription start sites in RNA from liver of adult mice. Transient transfection experiments revealed that 2.0 kilobase pairs of the L2 5'-flanking sequence exhibited promoter activity in BNL CL.2 (mouse liver) cells, CV-1 (monkey kidney) cells, and HRP.1 trophoblasts. Deletional analysis localized a major regulatory region to within 75 base pairs of the 5' transcription start site. Sequence analysis revealed that the region contained consensus binding sites for the Sp family of transcription factors. Standard gel shift and supershift analysis using liver nuclear extracts established that Sp1 and Sp3 bound this regulatory element. Transfection of wild type but not mutant decoy oligonucleotides into BNL CL.2 cells decreased the activity of the L2 promoter. Overexpression of Sp1 and Sp3 protein in Drosophila Schneider cells established that Sp3 is more potent than Sp1 in transactivating the L2 promoter. Co-transfection experiments further established that Sp1 antagonizes the activity of Sp3 to transactivate the L2 promoter. Western blot analysis of liver nuclear extracts revealed that the levels of Sp3 increase significantly after birth, suggesting a role for the Sp family of transcription factors in controlling the fetal to postnatal increase in GH receptor gene expression.

HIKE, a candidate protein binding site for PH domains, is a major regulatory region of G proteins

HIKE is a highly conserved sequence motif that selectively occurs in proteins candidate to bind PH domains, e.g., the β subunit of heterotrimeric G proteins, kinases, ankyrin and kinesin. Thus, the HIKE region has been predicted to be a protein docking site for PH domains. This work evidentiates recent experimental evidence that unambiguously defines the functional role of HIKE in Gβ as a multiple effector docking site and as a major regulatory region of G protein's function. Indeed, the Gβ HIKE interacts with the β-adrenergic receptor kinase, Gα, Gγ, adenylyl cyclase 2, phospholipase C β2, inward rectifier K channels, calcium channel α1B, calmodulin, phosducin, ste20. Quite interestingly, HIKE is located in the Gβ region that faces the cell membrane. Thus, HIKE also interacts with the cell membrane and may dynamically regulate membrane vs effector binding of the Gαβγ trimer. These findings fulfill a major prediction of the HIKE model, i.e., that HIKE is a regulatory region for protein-protein interactions. A role of HIKE as a proteic binding site for PH domains is supported by the profound influence of HIKE mutations on the largely PH-mediated binding of β-ARK to Gβ. Proteins 1999;35:360–363. © 1999 Wiley-Liss, Inc.

HIKE, a candidate protein binding site for PH domains, is a major regulatory region of G? proteins

HIKE is a highly conserved sequence motif that selectively occurs in proteins candidate to bind PH domains, e.g., the beta subunit of heterotrimeric G proteins, kinases, ankyrin and kinesin. Thus, the HIKE region has been predicted to be a protein docking site for PH domains. This work evidentiates recent experimental evidence that unambiguously defines the functional role of HIKE in Gbeta as a multiple effector docking site and as a major regulatory region of G protein's function. Indeed, the Gbeta HIKE interacts with the beta-adrenergic receptor kinase, Galpha, Ggamma, adenylyl cyclase 2, phospholipase C beta2, inward rectifier K channels, calcium channel alpha1B, calmodulin, phosducin, ste20. Quite interestingly, HIKE is located in the Gbeta region that faces the cell membrane. Thus, HIKE also interacts with the cell membrane and may dynamically regulate membrane vs effector binding of the Galphabetagamma trimer. These findings fulfill a major prediction of the HIKE model, i.e., that HIKE is a regulatory region for protein-protein interactions. A role of HIKE as a proteic binding site for PH domains is supported by the profound influence of HIKE mutations on the largely PH-mediated binding of beta-ARK to Gbeta.

Human endothelin-converting enzyme-1 beta mRNA expression is regulated by an alternative promoter.

The central step in endothelin biosynthesis is site-specific cleavage of big endothelins by endothelin-converting enzymes (ECEs). ECE-1 is a membrane-bound metalloprotease, predominantly but not exclusively expressed in endothelial cells. ECE-1 is expressed in two mRNA isoforms, termed alpha and beta, which differ only in the 5'-terminal regions but are functionally very similar when expressed in vitro. The structure of the human ECE-1 gene suggests either alternative splicing or alternative promoters as underlying mechanisms of mRNA isoform expression. We have previously shown that the alpha-upstream region exerts promoter activity in endothelial cells. To clarify whether the 5'-untranslated region upstream of exon 3, which contains the beta-specific sequence, acts as an alternative transcriptional promoter, we sequenced and cloned 1,206 bp upstream of the beta-specific translation initiation codon in a luciferase reporter vector. After transfection, we detected strong promoter activity in primary cultured endothelial cells (HU-VECs, BAECs) but only marginal activity in the endothelial cell line ECV304 and in CHO cells. Maximal promoter activity was observed with the full-length construct, 1206 (136% of the SV40 promoter activity in BAECs). Transfection of serial deletion mutants indicated at least three major regulatory regions within the promoter. Our results are consistent with cell type-restricted action of the beta-promoter and, in conjunction with the previously reported transcriptional start sites, clearly prove the existence of an alternative beta-specific promoter located in intron 2 of the human ECE-1 gene.

The tcp gene cluster of Vibrio cholerae.

The toxin co-regulated pilus (TCP) has been identified as a critical colonization factor in both animal models and humans for Vibrio cholerae O1. The major pilin subunit, TcpA (and also TcpB), is similar to type-4 pilins but TCP probably more appropriately belongs to a sub-class which includes the bundle-forming pilus of enteropathogenic Escherichia coli. The genes for TCP biosynthesis and assembly are clustered with the exception of housekeeping functions such as TcpG (=DsbA, a periplasmic disulfide bond epimerase). The nt sequences from El Tor and classical strains show only minor differences corresponding to the major regulatory regions and in TcpA itself. These differences are thought to account for the alternate conditions required for expression of TCP by the two biotypes and the antigenic variation and lack of cross-protection. Aside from the TcpA only a few of the proteins have had their roles in TCP biogenesis defined. Regulation of TCP is controlled by the ToxR regulon via ToxT with a possible involvement of TcpP and the cAMP-CRP system. Experiments using the infant mouse cholera model have now shown that TCP is a colonization factor and protective antigen for both classical and El Tor O1 strains and in the O139 Bengal serotype and that the mannose-sensitive haemagglutinin pilus does not appear to play a comparable role.

Comparison of the promoter proximal regions of the toxin-co-regulated tcp gene cluster in classical and E1 Tor strains of Vibrio cholerae O1

A physical map has been constructed of the 5-kb XbaI fragment encoding the promoter proximal of region the tcp gene cluster encoding the toxin-coregulated pilus (TCP) of Vibrio cholerae. This fragment contains the major regulatory regions for TCP. Comparison of the nucleotide (nt) sequences from strains of the classical and E1 Tor biotypes demonstrates that the regions are essentially identical, with several notable exceptions. The intergenic regions, between tcpI and tcpP, and between tcpH and tcpA, show significant sequence divergence which may account for the biotype-related differences in TCP, since this is the location of the major promoter sequences. The C-terminal coding regions of the major pilin subunit, TcpA, also differ. Southern hybridization analyses suggest that the tcpA nt sequence is conserved within a biotype, and Western blot analysis suggests that the two forms of TcpA are antigenically different, but related. Besides tcpA, tcpB, tcpH and tcpI, the genes encoding two additional proteins, TcpP and TcpQ, but not previously defined, were also identified. TcpH and TcpI have been previously suggested to be regulatory proteins but homology data imply that TcpI is a methyl-accepting chemotaxis protein (MCP), as recently reported [Harkey et al., Infect. Immun. 62 (1994) 2669-2678], and TcpH is predicted to be a periplasmic or exported protein. TcpP is thought to be a trans-cytoplasmic membrane (CM) protein which may have a regulatory role