The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death.

A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination. The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter. Mice homozygous for inactivated Nphs1 were born at an expected frequency of 25%. Although seemingly normal at birth, they immediately developed massive proteinuria and edema and died within 24 h. The kidneys of null mice exhibited enlarged Bowman's spaces, dilated tubuli, effacement of podocyte foot processes and absence of the slit diaphragm, essentially as found in human NPHS1 patients. In addition to expression in glomerular podocytes, the reporter gene was expressed in the brain and pancreas of (+/-) and (-/-) mice. In the brain, expression was localized to the ventricular zone of the fourth ventricle, the developing spinal cord, cerebellum, hippocampus and olfactory bulb. In the cerebellum, the expression was seen in radial glial cells. Neither anatomical nor morphological abnormalities were observed in the brains of null mice.

Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I.

Membrane-type matrix metalloproteinase I (MT1-MMP)-deficient mice were found to have severe defects in skeletal development and angiogenesis. The craniofacial, axial, and appendicular skeletons were severely affected, leading to a short and domed skull, marked deceleration of postnatal growth, and death by 3 wk of age. Shortening of bones is a consequence of decreased chondrocyte proliferation in the proliferative zone of the growth plates. Defective vascular invasion of cartilage leads to enlargement of hypertrophic zones of growth plates and delayed formation of secondary ossification centers in long bones. In an in vivo corneal angiogenesis assay, null mice did not have angiogenic response to implanted FGF-2, suggesting that the defect in angiogenesis is not restricted to cartilage alone. In tissues from null mice, activation of latent matrix metalloproteinase 2 was deficient, suggesting that MT1-MMP is essential for its activation in vivo.

Directional regulatory activity of cis-acting elements in the bidirectional alpha 1(IV) and alpha 2(IV) collagen gene promoter.

The genes for the type IV collagen alpha 1 and alpha 2 chains are regulated by a common alpha 130-base pair (bp) bidirectional promoter. In an attempt to explore the regulation of expression of both genes, we have constructed a minilocus plasmid containing minigenes for the human alpha 1 (IV) and alpha 2(IV) collagen genes located head-to-head like the endogenous genes. The directional regulation of this promoter was studied by introducing site-directed mutations into the promoter of the minigenes. In transient transfections the levels of transcripts of both minigenes were measured, and the influence of mutations on the direction of transcription was studied. The mutational analysis showed that a CCCTCCC motif and a GC box located in the center of the common promoter as well as GC boxes in exon 1 of the alpha 2(IV) gene are potent activating elements for both genes. The promoter of the alpha 1(IV) and alpha 2(IV) collagen genes contains no classical TATA boxes. Even so, A+T-rich sequence motifs, which have no obvious homology to the TATA box located 25-30 bp upstream of exon 1 in each gene, direct the initiation of transcription predominantly about 30 bp downstream of their location. Disruption of the A+T-rich sequence decreased the activity of the promoter considerably, and in addition, the generated classical TATA boxes hindered maintenance of normal bidirectional transcription and changed the ratio of the alpha 1(IV) and alpha 2(IV) transcripts. The CCAAT box in the alpha 2(IV) chain coding strand activated the promoter in the alpha 2(IV) gene direction, but not in the alpha 1(IV) gene direction.

The mouse Enhancer trap locus 1 (Etl-1): a novel mammalian gene related to Drosophila and yeast transcriptional regulator genes.

A novel mouse gene, Enhancer trap locus 1 (Etl-1), was identified in close proximity to a lacZ enhancer trap integration in the mouse genome showing a specific beta-galactosidase staining pattern during development. In situ analysis revealed a widespread but not ubiquitous expression of Etl-1 throughout development with particularly high levels in the central nervous system and epithelial cells. The amino acid sequence of the Etl-1 protein deduced from the cDNA shows strong similarity, over a stretch of 500 amino acids, to the Drosophila brahma protein involved in the regulation of homeotic genes and to the yeast transcriptional activator protein SNF2/SWI2 as well as to the RAD54 protein and the recently described helicase-related yeast proteins STH1 and MOT1. Etl-1 is the first mammalian member of this group of proteins that are implicated in gene regulation and/or influencing chromatin structure. The homology to the regulatory proteins SNF2/SWI2 and brahma and the expression pattern during embryogenesis suggest that Etl-1 protein might be involved in gene regulating pathways during mouse development.

Enhancer trap integrations in mouse embryonic stem cells give rise to staining patterns in chimaeric embryos with a high frequency and detect endogenous genes.

We have generated mouse embryonic stem cell lines that carry lacZ enhancer trap constructs integrated in their genome. Fifty-nine cell lines were analysed for lacZ expression in undifferentiated stem cells and at day 7.5, 8.5 and 12.5 of development in chimaeric embryos obtained after blastocyst injection. In 13 cell lines the lacZ reporter gene was expressed in undifferentiated stem cells ('blue', lines) as monitored by beta-galactosidase activity; 46 cell lines did not show detectable beta-galactosidase activity ('white', lines). In chimaeric embryos one-third of the analysed 59 embryonic stem cell lines gave rise to a variety of patterns. Six out of the 13 'blue' lines and 14 out of the 46 'white' lines showed spatially and temporally regulated patterns of beta-galactosidase expression and were additionally analysed on day 9.5. The majority of patterns showed staining exclusively or predominantly in structures of the developing nervous system, three patterns were observed only or predominantly in non-neuronal structures and five patterns were found exclusively in extraembryonic tissues. The analysis of DNA from cell lines that gave rise to staining patterns in chimaeric embryos showed that in 11 out of 15 cases simple integrations had occurred at a single site while in the remaining four cell lines multiple copies had integrated either at a single or at multiple sites. Flanking sequences from five reporter gene integrations have been cloned. At present, three integration sites have been analysed further and in all three cases we have identified transcribed sequences in the flanking DNA and isolated corresponding cDNA clones. The expression patterns of two of these genes were analysed by RNA in situ hybridisation. In both cases, expression of the endogenous genes was more widespread than the corresponding beta-galactosidase staining, suggesting that the reporter gene responded to only a subset of the regulatory elements of the endogenous gene. Our results demonstrate that enhancer trap integrations in embryonic stem cells can be used to efficiently identify transcriptional activation patterns during mouse embryogenesis and to isolate endogenous genes expressed in spatially and temporally regulated patterns.

Structural organization of the gene for the alpha 1 chain of human type IV collagen.

The complete exon size and distribution pattern in the gene for the alpha 1 chain of human type IV collagen was determined. Clones covering 145 kilobases (kb) of genomic DNA including 100 kb of the gene itself as well as 25 kb upstream and 20 kb downstream of the gene sequences, respectively, were isolated from lambda phage and cosmid libraries. The overall gene structure was determined by endonuclease restriction mapping and R-loop analyses and all exon sizes by nucleotide sequencing. The characterized clones contained all the coding sequences except for exon 2 whose sequence was determined after its amplification by the polymerase chain reaction. There were four gaps in the intron sequences; the exact size of the gene is unknown. The entire gene is at least 100 kb in size and contains 52 exons whose size distribution is completely different from that of the genes for fibrillar collagens. In the -Gly-X-Y- coding region there are three exons of 99, 90, and 45 base pairs (bp) each and two exons of 27, 36, 42, 51, 54, 63, and 84 bp each. The rest of the exons have sizes between 71 and 192 bp in the collagenous region. About one-half of the -Gly-X-Y- repeat coding exons start with the second base for the codon of glycine, whereas the other half starts (with two exceptions) with a complete glycine codon. The distribution of split versus unsplit codons is uneven in that the first 19 exons of the gene start with a complete codon. The gene contains repetitive sequences in several regions. A 185-nucleotide segment containing 40 copies of CCT flanked by poly(C) and poly(T) sequences was shown to be located adjacent to an exon. The gene has previously been shown to be located head-to-head to the alpha 2(IV) collagen gene at the distal end of the long arm of chromosome 13, such that the first exons of the two genes are separated by as little as 42 bp (Pöschl, E., Pollner, R., and Kühn, K. (1988) EMBOJ. 7,2687-2695; Soininen, R., Huotari, M., Hostikka, S. L., Prockop, D. J., and Tryggvason, K. (1988) J. Biol. Chem. 263, 17217-17220). The results demonstrate that the human alpha 1(IV) collagen gene has a structure distinctly different from the genes for fibrillar collagens and also that it is considerably larger than any collagen gene characterized to date.

The structural genes for alpha 1 and alpha 2 chains of human type IV collagen are divergently encoded on opposite DNA strands and have an overlapping promoter region.

Many of the genes of simple organisms with small genomes are encoded on opposite DNA strands so that the genes either overlap or one gene is nested within another gene (Normark, S., Bergström, S., Edlund, T., Grundström, T., Jaurin, B., Lindberg, F.P., and Olsson, D. (1983) Annu. Rev. Genet. 17, 499-525; Chen, C., Malone, T., Beckendorf, S.K., and Davis, R.L. Nature (1987) 329, 721-724). In contrast, most of the genes of complex organisms are dispersed in the genome in widely separated locations. Here, we report that the genes for the alpha 1 and alpha 2 chains of human type IV collagen are encoded on opposite DNA strands from loci that are so closely located that they may be separated by as little as 42 base pairs. The results provide the first description of two structural genes from a complex organism that code for two polypeptide chains of the same protein molecule but have overlapping 5'-flanking regions.

Complete primary structure of the alpha 1-chain of human basement membrane (type IV) collagen.

We have determined the primary structure of the alpha 1(IV)-chain of human type IV collagen by nucleotide sequencing of overlapping cDNA clones that were isolated from a human placental cDNA library. The present data provide the sequence of 295 amino acids not previously determined. Altogether, the alpha 1(IV)-chain contains 1642 amino acids and has a molecular mass of 157625 Da. There are 1413 residues in the collagenous domain and 229 amino acids in the carboxy-terminal globular domain. The human alpha 1(IV)-chain contains a total of 21 interruptions in the collagenous Gly-X-Y repeat sequence. These interruptions vary in length between two and eleven residues. The alpha 1(IV)-chain contains four cysteine residues in the triple-helical domain, four cysteines in the 15-residue long noncollagenous sequence at the amino-terminus and 12 cysteines in the carboxy-terminal NC-domain.

The gene for the alpha 1(IV) chain of human type IV procollagen: the exon structures do not coincide with the two structural subdomains in the globular carboxy-terminus of the protein.

Previous studies on the coding sequences of DNAs for the alpha 1(IV) chain of basement membrane collagen demonstrated a striking homology between the first 115 and the second 114 amino acids of the globular (NC1) domain of the protein. Also, alignment of the 12 cysteine residues indicated that the homology was particularly strong around three paired clusters of amino acids around cysteine residues. Here we have isolated a cosmid clone containing the 3'-end of the gene. Analysis of the clone and previously isolated lambda clones demonstrated that the intron--exon patterns of the gene does not reflect the homology in the protein. Therefore the homology cannot have arisen in any simple manner from gene duplications.

Large introns in the 3' end of the gene for the pro alpha 1 (IV) chain of human basement membrane collagen.

Using a recently characterized cDNA clone (HT-21) coding for the pro alpha 1 (IV) chain of human type IV procollagen, we have isolated three clones from a bacterio-phage lambda Charon 4A library of human genomic DNA. The intron/exon structure of the pro alpha 1 (IV) genomic clones was analyzed by heteroduplex electron microscopy and nucleotide sequencing. The analysis showed that the introns separating exons 2-9 are large and have a total length of over 12,000 base pairs (bp). Six of seven exons at the 3' end of the gene coded for -Gly-Xaa-Yaa-repeats of the collagenous part of the chain. Five of the -Gly-Xaa-Yaa- coding exons (numbers 5-9) varied in size between 72 bp and 134 bp, and none of them were 54 bp or multiples thereof. A sixth exon (exon 4) was a junction exon containing 71 bp coding for -Gly-Xaa-Yaa- sequences and 142 bp coding for the carboxyl-terminal noncollagenous domain (NC-1). The seventh exon (exon 3, 178 bp) coded for sequences of the NC-1 domain. Five of the six -Gly-Xaa-Yaa- coding exons began with the second base coding for glycine, and only one exon began with a complete glycine codon at the 5' end. The results (i) suggest that the gene for the pro alpha 1(IV) chain of human basement membrane collagen is significantly larger than the genes for fibrillar collagens and (ii) show that it lacks the 54-bp exon repeats characteristic of fibrillar collagen genes.

Circular dichroism studies on cytochrome c peroxidase and cytochrome c-551 of Pseudomonas aeruginosa.

Circular dichroism (CD) spectra of ferric, ferrous and ferrous-carbonyl forms of Pseudomonas cytochrome c peroxidase have been recorded in the wave length range 200 to 650 nm. CD spectra in the Soret region show that in the oxidized enzyme the two hemes are degenerate, whereas in the reduced form the hemes are perturbed differently and one of the hemes appears to be non-degenerate. Changes in optical activity upon formation of the carbonylderivative suggest a spin-state conversion and indicate the presence of one high-spin and a low-spin heme. A histidine residue is proposed for the axial ligand of the heme iron. The alpha-helical content of the enzyme is estimated to be 34%. Ligand binding or changes in the oxidation state of the heme iron do not alter the conformation of the protein backbone. The dichroic spectra of oxidized and reduced cytochrome c-551 (P. aeruginosa) are included for comparison. In the visible region the cytochrome exhibits CD spectra similar to those of the peroxidase, whereas in the Soret region the dichroic spectra of the cytochrome are simpler. CD spectra in the far-ultraviolet region show the cytochrome to have a high alpha-helix content.

Pseudomonas cytochrome c peroxidase. XIII. pH-denaturation of the enzyme.

The effect of pH on the oxidized Pseudomonas cytochrome c peroxidase molecule was studied by measuring the peroxidatic activity, the sedimentation velocity, the circular dichroic spectra in the far UV and Soret regions, and the optical absorption spectra of the enzyme in the pH range 2.5-13.0 at a constant ionic strength (micron = 0.1). The enzyme was stable in a narrow pH region, pH 6.0 - 7.4. In the low pH range the gross tertiary structure was observed to change quite simultaneously with the enzymatic activity and secondary structure. The optical absorption spectra indicated that there were no coordinated internal protein liqands in the 6th coordination positions of the heme prosthetic groups at the lowest pH studied. In the high pH range the secondary structure and the protein environment of hemes were observed to remain stable after the tertiary structure had changed and the activity had decreased. According to the optical absorption spectra the 6th internal protein ligands of hemes were retained at the highest pH studied.