Browsing by Author "Parker, M Iqbal"

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    The 341C/T polymorphism in the GSTP1 gene is associated with increased risk of oesophageal cancer
    (BioMed Central Ltd, 2010) Li, Dongping; Dandara, Collet; Parker, M Iqbal
    BACKGROUND: The Glutathione S-transferases (GSTs) comprise a group of enzymes that are critical in the detoxification of carcinogens. In this study the effects of polymorphisms in these genes on the risk of developing oesophageal squamous cell carcinoma (OSCC) were evaluated in a hospital-based case-control study in two South African population groups. Genetic polymorphisms in GSTs were investigated in 245 patients and 288 controls samples by PCR-RFLP analysis. RESULTS: The GSTP1 341T variant was associated with significantly increased risk of developing OSCC as observed from the odds ratios for the GSTP1 341C/T and GSTP1 341T/T genotypes (OR = 4.98; 95%CI 3.05-8.11 and OR = 10.9; 95%CI 2.43-49.1, respectively) when compared to the homozygous GSTP1 341C/C genotype. The risk for OSCC in the combined GSTP1 341C/T and T/T genotypes was higher in tobacco smokers (OR = 7.51, 95% CI 3.82-14.7), alcohol consumers (OR = 15.3, 95% CI 1.81-12.9) and those using wood or charcoal for cooking and heating (OR = 12.1, 95% CI 3.26-49) when compared to those who did not smoke tobacco, or did not consume alcohol or user other forms of fuel for cooking and heating. Despite the close proximity of the two GSTP1 SNPs (313A>G and 341C>T), they were not in linkage disequilibrium in these two population groups (D':1.0, LOD: 0.52, r2: 0.225). The GSTP1 313A/G polymorphism on the other hand, did not display any association with OSSC. The homozygous GSTT1*0 genotype was associated with increased risk of OSCC (OR = 1.71, 95%CI 1.18-2.46) while the homozygous GSTM1*0 genotype was associated with significantly decreased risk of OSCC in the Mixed Ancestry subjects (OR= 0.39, 95%CI 0.25-0.62). CONCLUSIONS: This study shows that the risk of developing OSCC in the South African population can be partly explained by genetic polymorphisms in GST coding genes and their interaction with environmental factors such as tobacco smoke and alcohol consumption.
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    Characterisation of the human α2(I) procollagen promoter-binding proteins
    (1993) Collins, Malcolm Robert; Parker, M Iqbal
    In an attempt to elucidate the transcriptional mechanisms that regulate the expression of the human α2(I) procollagen gene, cis-acting DNA-elements within the proximal promoter were identified and their corresponding trans-acting factors characterised. The fibroblast cell lines used in this study had previously been transformed with either simian virus 40 (SVWI-38) or by γ-radiation (CT-1). The SVWI-38 fibroblasts do not produce any α2(I) collagen chains, whereas the CT-1 cell line produces normal type I collagen. Previous studies suggested that trans-acting factor(s) may be responsible for the inactivation of the α2(I) procollagen gene in SVWI-38 fibroblasts (Parker et. al. (1989) J. Biol. Chem 264, 7147-7152; Parker et. al. (1992) Nucleic Acids Res. 20, 5825-5830). In this study, the SVWI-38 proximal promoter (-350 to +54) was sequenced and shown to be normal, thereby ruling out any possibility that mutations within this region was responsible for inactivation of the gene.
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    Collagen gene expression in human cancer
    (1997) Fenhalls, Gael; Parker, M Iqbal
    Type I collagen is the predominant collagen within the stroma and plays an important role in the processes of tumour cell invasion and metastasis during which the collagens within the stroma is degraded. Total RNA was extracted from different stages of breast cancer and adjacent normal tissue for analysis of collagen gene expression by Northern blot hybridisation. Stage I breast tumours had increased α1(I) and α2(I) collagen mRNA, whereas stages II and III tumours had decreased mRNA levels when compared to the adjacent normal tissue. This stage-specific change in collagen gene expression was confirmed by non-radioactive in situ hybridisation and the results indicated that α1(I) and α2(I) collagen mRNA was produced by the stromal fibroblasts and not the tumour cells. To determine whether this altered collagen gene expression was manifested in other cancers, α1(I) and α2(I) collagen mRNA levels were analysed in colorectal carcinoma samples by in situ hybridisation. Colon cancer as in the case of breast cancer, also showed stage specific changes in collagen gene expression. Dukes C and D colon cancer samples had decreased collagen mRNA levels compared to Dukes A and B. Mutated Ras has been shown to affect collagen mRNA levels in vitro (Slack et al, 1992), therefore the colon samples were analysed for Ras mutations in an attempt to correlate Ras mutations with the decreased levels of α1(I) and α2(I) collagen mRNA. Colorectal DNA samples were screened for Ras mutations by SSCP and direct sequence analysis. No possible association was found between the presence of Ras mutations and the decreased collagen gene expression. To gain greater insight into exactly how tumour cells modulate the collagen produced by normal fibroblasts, primary breast fibroblasts (prepared from breast tissue) were cocultured with various breast tumour cell lines. The fibroblasts were also incubated with conditioned media prepared from the tumour cells. Collagen production was analysed using the collagenase assay and the results showed that co-cultured tumour cells, as well as growth in the presence of tumour cell conditioned media, resulted in decreased type I collagen production by the fibroblasts. Type III collagen is often produced in conjunction with type I collagen and we have found that the breast tumour cells modulated type III collagen in the same way as type I collagen. These results demonstrated that a factor(s) was secreted by the tumour cells which affected collagen production. This factor was further shown to stimulate the fibroblasts to produce type I collagenase as analysis of the medium from co-cultured fibroblasts and tumour cells indicated the presence of collagenases. The tumour cell conditioned media was subsequently shown by Western blot analysis to contain a protein of similar molecular weight to the tumour cell derived collagenase stimulatory factor (known as EMMPRIN or extracellular matrix metalloproteinase inducer) which stimulates fibroblasts to secrete collagenases and has been shown to play a crucial role in tumour invasion (Biswas 1982, 1984 and Biswas et al, 1995). In order to determine whether fibroblasts of different origins reacted similarity when cocultured with breast tumour cell lines, WI-38 lung fibroblasts and FGo skin fibroblasts were co-cultured with breast tumour cells. WI-38 fibroblasts responded in the same way as breast fibroblasts (having decreased collagen production), FGo fibroblasts had no effect or slightly elevated collagen production, depending on the tumour cell line. These results suggested that the response to tumour cells is tissue specific. The decrease in type I collagen produced by the fibroblasts when incubated with the tumour cell conditioned media was not due to a decrease in α1(I) and α2(I) collagen mRNA as shown by Northern hybridisation. Type III collagen mRNA was affected differently, the levels were either decreased or increased depending on the tumour cell line being used. We postulate that the fibroblasts and tumour cells required contact for type I collagen mRNA to be decreased. Northern hybridisation showed that types I and III collagen mRNA levels were decreased when tumour cells were co-cultured with the fibroblasts. To demonstrate that specific contact was in fact required, the tumour cells were separated from the fibroblasts by a diffusible membrane and the levels of collagen mRNA were not adversely affected. Tumour cells, therefore can modulate collagen production by normal fibroblasts in two ways I); cause the fibroblasts to secrete collagenases which will degrade the collagen and 2); decrease collagen mRNA. Both of these mechanisms would aid the tumour in invasion and metastasis.
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    The cumulative effects of polymorphisms in the DNA mismatch repair genes and tobacco smoking in oesophageal cancer risk
    (Public Library of Science, 2012) Vogelsang, Matjaz; Wang, Yabing; Veber, Nika; Mwapagha, Lamech M; Parker, M Iqbal
    The DNA mismatch repair (MMR) enzymes repair errors in DNA that occur during normal DNA metabolism or are induced by certain cancer-contributing exposures. We assessed the association between 10 single-nucleotide polymorphisms (SNPs) in 5 MMR genes and oesophageal cancer risk in South Africans. Prior to genotyping, SNPs were selected from the HapMap database, based on their significantly different genotypic distributions between European ancestry populations and four HapMap populations of African origin. In the Mixed Ancestry group, the MSH3 rs26279 G/G versus A/A or A/G genotype was positively associated with cancer (OR = 2.71; 95% CI: 1.34-5.50). Similar associations were observed for PMS1 rs5742938 (GG versus AA or AG: OR = 1.73; 95% CI: 1.07-2.79) and MLH3 rs28756991 (AA or GA versus GG: OR = 2.07; 95% IC: 1.04-4.12). In Black individuals, however, no association between MMR polymorhisms and cancer risk was observed in individual SNP analysis. The interactions between MMR genes were evaluated using the model-based multifactor-dimensionality reduction approach, which showed a significant genetic interaction between SNPs in MSH2, MSH3 and PMS1 genes in Black and Mixed Ancestry subjects, respectively. The data also implies that pathogenesis of common polymorphisms in MMR genes is influenced by exposure to tobacco smoke. In conclusion, our findings suggest that common polymorphisms in MMR genes and/or their combined effects might be involved in the aetiology of oesophageal cancer.
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    DNA synthesis and methylation in normal and transformed cells
    (1985) De Haan, Judy Bettina; Parker, M Iqbal
    In this study, DNA methylation was examined during the eukaryotic cell cycle, and shown to occur throughout the S phase as well as during the "early" G₂ phase. However, DNA synthesis and methylation of newly synthesized DNA did not occur simultaneously, but the latter lagged behind DNA synthesis by about two hours. Once added during the S phase, the methyl groups were stably maintained in the DNA. Various compounds which are known to affect DNA synthesis in tissue cultured cells, were tested for their ability to alter the methylation status of DNA. The effects of three DNA synthesis inhibitors, viz. hydroxyurea (HU), 1-S-D-arabinofuranosyl cytosine (ara-C) and aphidicolin were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40 (SV 40) transformed line (SVWI-38) and a y-irradiation transformed cell line (CT-1). HU was shown to enhance hypermethylation of pre-existing DNA strands in the normal cells, while ara-C and aphidicolin caused hypermethylation of newly synthesized DNA strands. The effects of various concentrations of a known inducer of gene expression, sodium butyrate, were examined on these three cell lines as well. During a 16-20 hour treatment period, at butyrate concentrations of between 5 and 20 mM, no adverse effect on cell morphology was observed. Cell growth, in the presence of butyrate for 14 hours, showed that butyrate was more toxic on the transformed cells than on the normal cells. However, at 5 mM butyrate, DNA synthesis was inhibited by 75% in the normal cells, and was unaffected in the transformed lines. RNA synthesis was not affected in the transformed cells, whilst in the normal cell line, RNA synthesis was decreased to 76% of the control value, at sodium butyrate concentrations as low as 5 mM. Protein synthesis also was unaffected in the transformed cells and only slightly (+ 10%) inhibited in the normal cells at 20 mM butyrate. SDS polyacrylamide gel electrophoresis of proteins synthesized in the presence of 10 mM sodium butyrate, showed that most proteins were unaffected. Two high molecular weight proteins in the WI-38 cells appeared to be modified during butyrate. treatment, while one protein was induced by butyrate treatment in the CT-1 cells. More importantly though, butyrate treatment also resulted in hypermethylation of DNA, as shown by MSP 1 and Hpa II restriction endonuclease digestion and high-pressure liquid chromatography analysis. Butyrate appeared to specifically cause hypermethylation of pre-existing DNA strands in the WI-38 cells, while the SVWI-38 and CT-1 cells showed preferential hypermethylation of newly synthesized DNA strands. However, the hyper-methylated state was only heritable if the methylation event occurred in newly synthesized DNA. Hypermethylation on pre-existing DNA was rapidly lost in the subsequent generation. It would therefore appear that methylcytosines are only maintained in the DNA if they are generated on newly synthesized DNA. This study has clearly shown that the heritability of DNA methylation patterns is closely linked to DNA replication.
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    Gene mutations and expression in breast cancer
    (2000) Donninger, Howard; Parker, M Iqbal
    Breast cancer is the most common cause of death amongst women, with the incidence of the disease varying between countries. Like all other cancers, breast cancer is a multigenic disorder with mutations in oncogenes and tumour suppressor genes playing an important role in cellular transformation and ultimately in tumour formation. In this study, 40 breast cancer patients from the Western Cape province in South Africa and 4 breast cancer cell lines were screened for mutations in the human Ha-ras oncogene and the p53 tumour suppressor gene.
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    Identification of genetic polymorphisms associated with oesophageal squamous cell carcinoma risk in South Africa
    (2013) Matejcic, Marco; Parker, M Iqbal; Mathew, Chris
    Oesophageal squamous cell carcinoma (OSCC) is a complex disease, determined by the interaction of genetic factors with environmental risk factors. In South Africa, OSCC is a major malignancy occurring with high incidence in the Black and Mixed Ancestry populations. Previous studies by our research group have reported that genetic polymorphism of xenobiotic metabolizing enzymes influence greatly the detoxification of tobacco-related carcinogens in vivo, and may therefore have an important role in determining susceptibility to oesophageal cancer.
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    Investigation into the effects of the tobacco smoke procarcinogen benzo[a]pyrene on gene expression profiles in oesophageal cancer
    (2011) Bick, Alexis J; Parker, M Iqbal
    Tobacco smoking is a major risk factor in the development of oesophageal squamous cell carcinoma (OSCC). Benzo[a]pyrene (BaP) is a tobacco smoke procarcinogen that is metabolically activated into the carcinogenic benzo[a]pyrene diol-epoxide (BPDE) by the CYP1 family of cytochrome P450 enzymes. BaP is a ligand for the aryl hydrocarbon receptor (AhR) which activates CYP1 gene transcription. Polymorphisms in these genes affect enzyme activity and therefore BaP bioactivation.
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    Rat angiotensin-converting enzyme : tissue specific expression during pharmacological inhibition
    (1995) Brice, Edmund Andrew William; Kirsch, Ralph E; Parker, M Iqbal
    The renin-angiotensin system plays a central role in the maintenance of blood pressure. Angiotensin II, the main effector of this system, results from the action of angiotensin-converting enzyme (ACE) on angiotensin I. Angiotensin II, maintains vasomotor tone via its vasoconstrictor action, and also increases salt and water retention by stimulating the release of aldosterone. ACE inhibitors, such as captopril, enalapril and lisinopril, are highly effective in the treatment of hypertension and congestive cardiac failure. Previous studies have suggested that angiotensin converting enzyme (ACE) production may be enhanced during pharmacological inhibition of the enzyme. Little is known, however about the mechanism of this induction. After demonstrating increases in circulating ACE protein in cardiac failure patients receiving the ACE inhibitor captopril, a rat model was used to study this effect. A sensitive enzyme linked immunosorbent assay for rat ACE was developed and a partial cDNA for rat ACE cloned to enable examination of ACE mRNA and protein expression during enzyme inhibition with enalapril. Rat lung ACE mRNA increased by 156% (p<0.05) and ACE protein doubled within 3 hours of administering a single dose of enalapril. Testicular ACE mRNA also increased by 300% (p<0.05) within 2 hours and returned to pretreatment levels by 6 hours. The angiotensin II antagonist saralasin similarly caused a significant (p<0.0001) 800% enhancement of mRNA expression. Aldosterone pretreatment of rats prior to enalapril administration was found to abolish this mRNA induction. These findings indicate that increased ACE expression during inhibition results from reduced levels of angiotensin II with consequent reduced stimulation of the angiotensin 11 receptor and its effects, such as aldosterone release. This suggests that ACE levels are regulated by a negative feedback loop involving the distal components of the renin-angiotensin system, namely angiotensin II and aldosterone. In situ hybridisation and immunohistochemical techniques were employed to localise the site of this inductive response in rat tissue sections. It was found that lung macrophages were markedly induced to produce ACE, as was ACE in seminiferous tubules. ACE induction was also noted in the expected sites of renal tubular epithelium and glomerular tissue. Interestingly, ACE expression was also enhanced in cardiac valves. In these studies it has been conclusively demonstrated that new ACE expression is induced by enzyme inhibitor therapy. A variety of techniques have been developed that will allow futher study of ACE in rat tissues.
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    The role of DNA methylation in transcriptional regulation of the human type 1 alpha 2 collagen (COL1A2) gene
    (2002) Ndlovu, Matladi N; Parker, M Iqbal
    Type I collagen is the most abundant collagen molecule in vertebrate connective tissue and it consists of a heterotrimer of two alpha 1 (COL1A1) and one alpha 2 (COL1A2) chains. Reduced collagen gene expression is almost always correlated with pathological conditions and cellular transformation. Numerous studies have suggested that methylation of the cytosines in CpG dinucleotides is inversely correlated with transcriptional activity and plays a critical role in differential gene expression.
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    The role of human papillomavirus (HPV) E6 proteins as a risk factor for oesophageal cancer
    (2007) Ross-Innes, Caryn Sarah; Parker, M Iqbal; Dandara, Collet
    Oesophageal squamous cell carcinoma (OSCC) is a major cancer in South Africa, affecting mainly black males. Several risk factors for OSCC have been reported but this study focuses on the role of human papilloma virus (HPV) in the development of OSCC. HPV is a well-known risk factor for cervical cancer resulting in its classification into low- and high-risk HPV types. The role of the different HPV types in OSCC development is not known, but in cervical cancer the critical HPV transforming gene has been shown to be E6. In this project, the effects of HPV11 E6, a low-risk type, and HPV18 E6, a high-risk type, were investigated by transfecting HPV-negative cell lines (EPC2-hTERT, MCF12A and Rat1) with HPV11 and HPV18 E6.
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    The role of inflammation in HPV infection of the Oesophagus
    (BioMed Central Ltd, 2013) Schäfer, Georgia; Kabanda, Siti; van Rooyen, Beverly; Marusic, Martina Bergant; Banks, Lawrence; Parker, M Iqbal
    BACKGROUND: Several human cancers are known to be associated with inflammation and/or viral infections. However, the influence of tumour-related inflammation on viral uptake is largely unknown. In this study we used oesophageal squamous cell carcinoma (OSCC) as a model system since this type of cancer is associated with chronic irritation, inflammation and viral infections. Although still debated, the most important viral infection seems to be with Human Papillomavirus (HPV). The present study focused on a possible correlation between inflammation, OSCC development and the influence of HPV infection. METHODS: A total of 114 OSCC biopsies and corresponding normal tissue were collected at Groote Schuur Hospital and Tygerberg Hospital, Cape Town (South Africa), that were subjected to RNA and DNA isolation. RNA samples were analysed by quantitative Light Cycler RT-PCR for the expression of selected genes involved in inflammation and infection, while conventional PCR was performed on the DNA samples to assess the presence of integrated viral DNA. Further, an in vitro infection assay using HPV pseudovirions was established to study the influence of inflammation on viral infectivity using selected cell lines. RESULTS: HPV DNA was found in about 9% of OSCC patients, comprising predominantly the oncogenic type HPV18. The inflammatory markers IL6 and IL8 as well as the potential HPV receptor ITGA6 were significantly elevated while IL12A was downregulated in the tumour tissues. However, none of these genes were expressed in a virus-dependent manner. When inflammation was mimicked with various inflammatory stimulants such as benzo-alpha-pyrene, lipopolysaccharide and peptidoglycan in oesophageal epithelial cell lines in vitro, HPV18 pseudovirion uptake was enhanced only in the benzo-alpha-pyrene treated cells. Interestingly, HPV pseudovirion infectivity was independent of the presence of the ITGA6 receptor on the surface of the tested cells. CONCLUSION: This study showed that although the carcinogen benzo-alpha-pyrene facilitated HPV pseudovirion uptake into cells in culture, HPV infectivity was independent of inflammation and seems to play only a minor role in oesophageal cancer.
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    Screening of variants for lactase persistence/non-persistence in populations from South Africa and Ghana
    (BioMed Central Ltd, 2009) Torniainen, Suvi; Parker, M Iqbal; Holmberg, Ville; Lahtela, Elisa; Dandara, Collet; Jarvela, Irma
    BACKGROUND:Lactase non-persistence is a condition where lactase activity is decreased in the intestinal wall after weaning. In European derived populations a single nucleotide polymorphism (SNP) C/T-13910 residing 13.9 kb upstream from the lactase gene has been shown to define lactase activity, and several other single nucleotide polymorphisms (G/C-14010 T/G-13915, C/G-13907 and T/C-13913) in the same region have been identified in African and Middle East populations. RESULTS: The T-13910 allele most common in European populations was present in 21.8% mixed ancestry (N = 62) individuals and it was absent in the Xhosa (N = 109) and Ghana (N = 196) subjects. Five other substitutions were also found in the region covering the previously reported variants in African and Middle East populations. These included the G/C-14010 variant common in Kenyan and Tanzanian populations, which was present in 12.8% of Xhosa population and in 8.1% of mixed ancestry subjects. Two novel substitutions (C/T-14091 and A/C-14176) and one previously reported substitution G/A-13937 (rs4988234) were less common and present only in the Xhosa population. One novel substitution G/A-14107 was present in the Xhosa and Ghanaian populations. None of the other previously reported variants were identified. CONCLUSION: Identification of the G/C-14010 variant in the Xhosa population, further confirms their genetic relatedness to other nomadic populations members that belong to the Bantu linguistic group in Tanzania and Kenya. Further studies are needed to confirm the possible relationship of the novel substitutions to the lactase persistence trait.
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    Transcriptional regulation of the human alpha 2(I) procollagen gene
    (1997) Leaner, Virna Drucille; Parker, M Iqbal
    The objective of this study was to investigate the cell- and species-specific regulation of the α2(1) pro collagen gene by analysing trans-acting factor interactions within the proximal promoter of the gene and to identify the genes coding for these trans-acting factors. α2(1) procollagen gene expression was examined in a number of diff erentiate<;l cell lines and shown to differ significantly between normal fibroblasts (WI-38, FG₀), transformed fibroblasts (CT-1, SVWI-38), HT1080 fibrosarcoma, HepG2 hepatocellular carcinoma, L77 lymphoblasts and breast cancer epithelial cells (MDA-MB-231, ZR-75-2). These differences were due to changes in transcription of the α2(1) procollagen gene as shown by Northern blot analysis and nuclear runon transcription experiments . Analysis of DNA-protein interactions with the proximal α2(1) procollagen promoter showed the presence of at least two DNA-protein complexes (complexes I and III) in collagen producing cell lines, while cells where collagen synthesis did not occur contained a third DNA-protein complex (complex II). α2(1) procollagen gene expression was therefore shown to be associated with the presence of complexes I and III while repression of the gene was associated with the presence of complexes I and II and the partial or complete absence of complex III. Complex I is a ubiquitous factor which binds the inverted CCAAT box located between -92 and -80 (G/CBE) with an apparent Kd of 2.9nM. Complexes II and III both bind an adjacent DNA sequence between -78 and -67 (the CME) with Kd values of 4.2 and 3.5nM respectively. While the CCAA T boxes in the human and mouse promoters are identical, a 3bp mismatch was detected in the CME. This mismatch abolished the formation of complex II and III on the mouse promoter, even though mouse cells contained complex II proteins. The difference in the CME binding site between rodent and human promoters implied species-specific regulation of the α2(1) procollagen gene. Transfection of human and mouse proximal α2(1) procollagen promoter/CAT constructs into human cells (CT-1) indicated that the human promoter had higher activity than the mouse promoter, whilst the two promoters had equivalent activities in rodent cells. These promoter activities may be accounted for by the differences in trans-acting factor binding to the two promoters. Complex I formation was competed out by the mouse CBF and NF-Y consensus oligonucleotides, while the mouse anti-CBF-B antibody resulted in a supershifted complex I. These results indicate that complex I is a member of the heterologous CCAAT-binding proteins and possibly related to or similar to the mouse CBF. The treatment of nuclear extracts with calf intestinal phosphatase resulted in a loss of complex I formation on the human and CBF binding to the mouse promoters. The Ser/Thr phosphatase, PP2A, specifically inhibited complexes II and ill formation. Nuclear extracts from CT-1 and U937 cell lines treated with the kinase inhibitor, staurosporin, was accompanied by a loss in DNA-protein interaction. This inhibition of DNA-binding activity was not observed using the tyrosine kinase inhibitor, genistein, and the PP2A phosphatase inhibitor, okadaic acid. Staurosporin also had a significant inhibitory effect on α2(1) procollagen promoter activity in CT-1 cells transfected with the human proximal α2(1) procollagen promoter and on steady state collagen mRNA levels. These results indicate that phosphorylation is required for the binding of trans-acting factors to the proximal α2(1) procollagen promoter and in transcriptional regulation of this gene. In support of the suggestion that phosphorylation events play a role in transcriptional regulation of the α2(1) procollagen gene, CT-1 cells treated with the protein kinase C activator, PMA, showed a significant reduction in α2(1) procollagen mRNA levels. A lambda gt11 expression library was screened to obtain cDNA's encoding proteins that bind the CME in the human α2(1) proximal promoter. A cDNA clone of 958 bp with a predicted open reading frame of 116 amino acids (12.5kD) was obtained. No significant DNA or polypeptide sequence homologies existed in the databank, indicating the possibility of a novel trans-acting factor. Binding of this fusion protein was specific for the CME as observed in South Western blotting and gel shift assays using competitor DNA sequences. Northern blot analysis detected a mRNA transcript of approximately 4kb predominantly in cells where α2(1) procollagen expression is repressed.