Linda Wood

cDNA clones completing the nucleotide and derived amino acid sequence of the alpha 1 chain of basement membrane (type IV) collagen from mouse

Six cDNA clones add 3549 nucleotides to the DNA sequence of the alpha 1 chain of basement membrane (type IV) collagen. Thus the complete nucleotide and derived amino acid sequence of the alpha 1 type IV collagen with 5007 nucleotides coding for 1669 amino acids with a calculated M r of 160 827 is known. The six cDNA clones cover the putative N‐terminal signal peptide, the 7 S region and two thirds of the helical region extending into the previously published murine nucleotide sequence [(1986) Gene 43, 301]. The protein sequence for 289 amino acids of the helical region adjacent to the 7 S region has not previously been published for any species.

High-sulfur protein gene expression in a transgenic mouse.

We analyzed the effect of minoxidil on hair follicles isolated from transgenic mice. These transgenic animals synthesize the reporter enzyme CAT in their hair follicles only during the active phases of hair growth. The recombinant gene used to generate these mice contained the bacterial enzyme CAT under the control of the promoter from the gene of UHS protein. Studies using in situ hybridization showed that UHS proteins are expressed specifically in the matrix cells of the hair follicle during the terminal stages of hair differentiation. Hence the expression of the UHS proteins is a clear sign of active hair growth. With other in situ hybridization studies we demonstrated that CAT mRNA is expressed in differentiating matrix cells of the hair shaft in a location similar to that in which mRNA encodes UHS proteins. Thus we can use the levels of CAT activity as a measure of hair growth. We have confirmed that expression of the transgene is found in hair that is high in anagen and low in catagen follicles. The usefulness of our model was further demonstrated by showing that minoxidil, a drug that stimulates hair growth, increased the expression of CAT in cultured hair follicles. Thus we have demonstrated that expression of this reporter gene is sensitive, hair specific, and also useful for monitoring effects in cultured hair follicles. Hence these transgenic mice provide a model system for studying the biology of hair growth.

MOLECULAR CLONING OF THE BOVINE α1(IV) PROCOLLAGEN GENE (COL4A1) AND ITS USE IN INVESTIGATING THE REGULATION OF EXPRESSION OF TYPE IV PROCOLLAGEN BY RETINOIC ACID IN BOVINE LENS EPITHELIAL CELLS

This report is the first to describe the isolation of a 400 base pair cDNA clone encoding part of the bovine α1(IV) procollagen. Using the polymerase chain reaction (PCR), we have amplified a sequence of approximately 400bp from this gene within the recombinant phage DNA. The cloned sequence encodes 94 amino acids that form part of the proteins helical region. The sequence contains one interruption in the Gly‐Xaa‐Yaa repeat unit. The third base of the codon for glycine at several sites differs from those seen in murine and human genes, as does the third base of proline codons. The bovine cDNA also contains fewer thymine residues. Northern blot hybridization has shown that the mRNA for bovine procollagen to be 6.2kb in size. We have used the cDNA clone to investigate the effect of all‐transretinoic acid (RA) on the gene expression of α1(IV) procollagen in cultured bovine lens epithelial (LE) cells. We have also observed that RA decreases total protein production and concomitantly increases type IV procollagen in a concentration dependent manner. An increase in α1(IV)mRNA as well as increase in type IV procollagen suggest that the regulation of α1(IV) gene by RA in the LE cells is at the transcriptional level. Further, our results support the hypothesis that RA inhibition of lens epithelium transformation to fibroblast‐like cells may be due to the ability of RA to stimulate the production of basement membrane components by epithelia.