Anne Olsen

Transcription of Human Type I Collagen Genes. Variation in the Relative Rates of Transcription of the Proα1 and Proα2 Genes

Abstract The relative rates of transcription of the two genes for type I collagen have been measured in a runoff transcription assay using nuclei isolated from cultured human fibroblasts. Control experiments indicated that the ratio of proα(I)/proα(I) transcripts detected with a given nuclear preparation did not vary over a range of transcription times, nuclei concentrations, and amounts of filter-bound cDNA used in the assay. However, a significant difference in the ratio was observed when nuclei isolated from cells grown under different conditions were used. Nuclei from sub-confluent cultures generally transcribed the two genes in a ratio of 2:1 or lower, while nuclei from post-confluent cultures transcribed the genes in a much higher ratio of about 4:1. Analysis of the amount of proα(I) and proα(I) RNA transcribed suggests that it is the transcription of the proα(I) gene, and not the proα(I) gene, that varies with culture conditions. The steady state ratios of proα(I)/proα(I) RNA remained near 2:1 under all conditions. Thus, some post-transcriptional mechanism apparently results in maintenance of the steady-state proα(I)/proα(I) RNA ratios at approximately 2:1.

[4] Relative transcription rates and mRNA levels for the two chains of type I procollagen

Publisher Summary This chapter discusses the relative transcription rates and mRNA levels for the two chains of type I procollagen. Type I collagen, the most abundant connective tissue protein in skin and bone, is composed of two αl chains and one α2 chain. These chains are derived from larger precursor polypeptides called pro-αl and pro-α2. For accurate determination of mRNA ratios, the probes used should be of the same size and should represent portions of the two mRNAs with similar base sequence and composition, but should not cross-hybridize to any appreciable extent. These criteria are met by the experimental conditions described in this chapter. With this technique, the ratio of pro-α1/pro-α2 mRNA has been consistently found to be about 2:1 in near confluent cultures of normal human fibroblasts. However, in some of the osteogenesis imperfecta fibroblasts examined the ratios deviate significantly from 2 : 1,2 an observation suggesting the presence of mutations that alter either the transcription of one of the genes or the processing of RNA transcripts,

Regulation of α1 (I) collagen gene promoter by tumor necrosis factor-α in human dermal fibroblasts

The r01e of cy七okines in 七he regu1ation of connec七ive 七issue metabolism has been increasingly emphasized by e1ucida七ionof 七heir specific effec七s using recombinan七 geneproduc七s. Recent s七udies have demons七rated 七ha七七umornecrosis fac七or-α(TNF-α) decreases α1 (工) collagen gene expression in cultured human dermal fibroblas七s. The purpose of this s七udywas to ana1yze transcrip七ional control of the α1 (工) collagen gene by TNF-αby means of DNA mediated 七ransfectionexperimen七s using recombinant plasmids in which七he promoter region of七hehuman α1 (工) co11agen had been fused七o 七he chloramphenicol ace七yl七ransferase (CAT) gene, in human derma1 fibroblas七s. Human dermal fibroblas七 monolayer cultures were preincuba七ed for 24 hours wi七h recombinan七 TNF-αranging from 1-100 ngjm工 Cellswere transfec七edwith 20 Jlg of七hechimeric collagen CAT gene by 七he calcium phosphate technique. pBLCAT (七hymidine kinase promo七er fused to the CAT gene) was used as the con七rol. We measured 七he levels of CAT ac七ivi七y 48 hours af七ertransfec七ion. Approximately 5-fold decreased levels of CAT ac七ivi七y were observed in fibroblas七s treated with 1 ngjml of TNF-α, when 2300 bp of 七he α1 (工 collagen promo七er gene fused 七O 七he CAT gene was transfected. Similar resul七s were al so ob七ained in the fibroblasts, when bo七h 800 and 330 bp of 七heα1 (工) collagen promoter fused to 七he CAT gene were 七ransfected. On 七he 0七her hand, 七he 1evels of CAT activi七y were una工七ered in fibrob1as七S 七ransfec七ed wi七h 七he con七rol gene. Our da七a show 七ha七七he expression of chimeric collagen CAT gene is strongly inhibi七ed by TNF-αin human dermal fibroblas七s, sugges七ing that a common mechanism inhibi七sboth transfec七edand endogenous α1 (工) collagen promoters. 工七 is also sugges七ed七hatTNF-α reduces α1 (工) collagen 七ranscription七hrougha七 leas七 upto 330 bp upstream of the α1 (工) collagen promo七er. Work is in progress七o analyze regions further downstream to de七ermine which elemen七s are responsible for decreased transcrip七ionwi七h TNF-α.