Gabriel Vogeli

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.

Evolution of collagen IV genes from a 54-base pair exon: A role for introns in gene evolution

SummaryThe exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse α2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exon sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the α2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such “fusion exons” the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculate that this mechanism could provide a role for introns in gene evolution in general.

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.

Proposed alignment of helical interruptions in the two subunits of the basement membrane (type IV) collagen

We have isolated a cDNA clone for part of the α2 type IV collagen (pCIV‐2‐176). Deoxynucleotide sequence analysis shows that this clone codes for 439 amino acids from the helical domain adjacent to the C‐terminal globular domain of the α2 (IV) chain. By aligning the deduced amino acid sequence of the α2 (IV) chain with the published sequence for the α1 (IV) chain, we find that all interruptions in the α1 (IV) chain coincide with an interruption in the α2 (IV) chain. Additonal interruptions in the α2 (IV) chain exist, however, three out of the four analysed only slightly disturb the collagen triple helix.

Homologies between the non-collagenous C-terminal (NC1) globular domains of the α1 and α2 subunits of type-IV collagen

Abstract The non-collagenous C-terminal globular domain (NC1) of type-IV collagen has the dual role of initiating triple-helix formation among the subunits and of crosslinking two collagen molecules during basement-membrane meshwork formation. By cloning a cDNA for the NC1 domain of the α2(IV) collagen chain, we have found a high degree of homology (63% for nucleotides, 66% for amino acids) between the NC1 of the α2 and αl chains of type-IV collagen. All cysteine residues are conserved. This high degree of homology is not found within the helical portion where the homology is 41 % for amino acids (only 14% if the obligatory glycine is not used for this analysis). We propose that this high degree of homology within the non-collagenous domain indicates a close evolutionary relationship maintained by functional restraints between the two chains of type IV collagen.

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.