ABSTRACT
Type XIII collagen is a type II transmembrane protein found at many sites of cell adhesion in tissues. Homologous recombination was used to generate a transgenic mouse line (Col13a1(N/N)) that expresses N-terminally altered type XIII collagen molecules lacking the short cytosolic and transmembrane domains but retaining the large collagenous ectodomain. The mutant molecules were correctly transported to focal adhesions in cultured fibroblasts derived from the Col13a1(N/N) mice, but the cells showed decreased adhesion when plated on type IV collagen. These mice were viable and fertile, and in immunofluorescence stainings the mutant protein was located in adhesive tissue structures in the same manner as normal alpha1(XIII) chains. In immunoelectron microscopy of wild-type mice type XIII collagen was detected at the plasma membrane of skeletal muscle cells whereas in the mutant mice the protein was located in the adjacent extracellular matrix. Affected skeletal muscles showed abnormal myofibers with a fuzzy plasma membrane-basement membrane interphase along the muscle fiber and at the myotendinous junctions, disorganized myofilaments, and streaming of z-disks. The findings were progressive and the phenotype was aggravated by exercise. Thus type XIII collagen seems to participate in the linkage between muscle fiber and basement membrane, a function impaired by lack of the cytosolic and transmembrane domains.
Subject(s)
Cell Membrane/metabolism , Collagen Type XIII/metabolism , Cytosol/metabolism , Muscular Diseases/etiology , Protein Structure, Tertiary , Amino Acid Sequence/genetics , Animals , Cell Adhesion/physiology , Cells, Cultured , Collagen Type XIII/chemistry , Collagen Type XIII/genetics , Disease Progression , Exons , Fibroblasts/physiology , Gene Deletion , Mice , Mice, Transgenic , Microscopy, Immunoelectron , Molecular Sequence Data , Motor Activity , Muscle, Skeletal/pathology , Muscle, Skeletal/ultrastructure , Muscular Diseases/pathology , Muscular Diseases/physiopathology , Recombination, GeneticABSTRACT
Type XIII collagen is a type II transmembrane protein found at sites of cell adhesion. Transgenic mouse lines were generated by microinjection of a DNA construct directing the synthesis of truncated alpha1(XIII) chains. Shortened alpha 1(XIII) chains were synthesized by fibroblasts from mutant mice, and the lack of intracellular accumulation in immunofluorescent staining of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to fetal lethality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a lack of fusion of the chorionic and allantoic membranes. The late phenotype fetuses were aborted by day 13.5 of development and displayed a weak heartbeat, defects of the adherence junctions in the heart with detachment of myofilaments and abnormal staining for the adherence junction component cadherin. Decreased microvessel formation was observed in certain regions of the fetus and the placenta. These results indicate that type XIII collagen has an important role in certain adhesive interactions that are necessary for normal development.
Subject(s)
Adherens Junctions/ultrastructure , Collagen/genetics , Collagen/physiology , Heart Defects, Congenital/pathology , Neovascularization, Physiologic , Animals , Collagen/metabolism , Embryonic and Fetal Development , Fetus/abnormalities , Fetus/blood supply , Heart/embryology , Mice , Mice, Transgenic , Mutation , Myocardium/ultrastructure , Phenotype , Placenta/abnormalities , Placenta/blood supply , RNA, Messenger/biosynthesis , Survival AnalysisABSTRACT
Fluorescence in situ hybridization (FISH) on mechanically stretched chromosomes (MSCs) and extended DNA fibers enables construction of high-resolution physical maps by accurate ordering and orienting genomic clones as well as by measuring physical lengths of gaps and overlaps between them. These high-resolution FISH targets have hitherto been used mainly in the study of the human genome. Here we have applied both MSCs and extended DNA fibers to the physical mapping of the mouse genome. At first, five mouse collagen genes were localized by metaphase-FISH: Col10a1 to chromosomal bands 10B1-B3; Col13a1 to 10B4; and Col6a1, Col6a2, and Col18a1 to 10B5-C1. The mutual order of the genes, centromere--Col10a1--Col13a1--Col6a2--Col6a1--Col18a1--telomere, was determined by FISH on metaphase chromosomes, MSCs, and extended DNA fibers. To our knowledge, this is the first time mouse metaphase chromosomes have been stretched and used as targets for FISH. We also used MSCs to determine the transcriptional orientations, telomere--5'-->3'--centromere, of both Col13a1 and Col18a1. With fiber-FISH, Col18a1, Col6a1, and Col6a2 were shown to be in a head-to-tail configuration with respective intergenic distances of about 350 kb and 90 kb. Comparison of our physical mapping results with the homologous human data reveals both similarities and differences concerning the chromosomal distribution, order, transcriptional orientations, and intergenic distances of the collagen genes studied.
Subject(s)
Collagen/genetics , In Situ Hybridization, Fluorescence , Physical Chromosome Mapping , Animals , Gene Order/genetics , Mice , Multigene Family/genetics , Transcription, Genetic/geneticsABSTRACT
The recombinant transmembrane protein type XIII collagen is shown to reside on the plasma membrane of insect cells in a 'type II' orientation. Expressions of deletion constructs showed that sequences important for the association of three alpha1(XIII) chains reside in their N- rather than C-terminal portion. In particular, a deletion of residues 63-83 immediately adjacent to the transmembrane domain abolished the formation of disulfide-bonded trimers. The results imply that nucleation of the type XIII collagen triple helix occurs at the N-terminal region and that triple helix formation proceeds from the N- to the C-terminus, in opposite orientation to that of the fibrillar collagens. Interestingly, a sequence homologous to the deleted residues was found at the same plasma membrane-adjacent location in other collagenous transmembrane proteins, suggesting that it may be a conserved association domain. The type XIII collagen was secreted into insect cell medium in low amounts, but this secretion was markedly enhanced when the cytosolic portion was lacking. The cleavage occurred in the non-collagenous NC1 domain after four arginines and was inhibited by a furin protease inhibitor.
Subject(s)
Collagen/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Collagen/chemistry , Conserved Sequence , Disulfides/chemistry , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Furin , Insecta , Membrane Proteins/chemistry , Microscopy, Immunoelectron , Molecular Sequence Data , Protease Inhibitors/pharmacology , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, Protein , Subtilisins/antagonists & inhibitors , Subtilisins/metabolismABSTRACT
Recent findings indicate that type XIII collagen is a transmembrane protein with a short N-terminal sytocsolic domain, a single transmembrane domain and a large, mainly collagenous ectodomain. The complete exon-intron structure of the gene coding for the mouse alpha1(XIII) collagen chain, col13a1, has now been characterized from genomic clones spanning over 180 kilobases (kb) and shown to be approximately 135 kb in size and to contain 42 exons varying between 8 base pairs (bp), the shortest exon in the genes encoding the various collagens, and 836 bp. Nuclease S1 mapping and 5'RACE resulted in identification of multiple transcription initiation points in the mouse gene, ranging between 470 and 548 bp upstream from the initiation methionine. This is in good agreement with a recently identified human EST clone extending 537 bp upstream from the initiation methionine. The 836-bp first exon of the mouse gene covers both the long 5' untranslated region and also a 36-residue cytosolic portion, a 23-residue transmembrane domain, and 37 residues of the 60-residue non-collagenous ectodomain immediately adjacent to the plasma membrane. One striking feature of the exons encoding solely collagenous sequences is the abundance of 27-bp exons, half the ancestral 54-bp size characteristic of fibrillar collagen genes, while the others vary between 8 and 144 bp, including instances of 36-, 45- and 54-bp exons. Determination of approximately 2.6 kb of sequences upstream of the initiation methionine of both the mouse and human genes and the identification of a clone containing four exons and spanning a gap in the previously characterized human clones allowed detailed comparison of the two genes. The exon-intron structures were found to be completely conserved between the species, and both genes have their 5' untranslated region preceded by a highly homologous apparent promoter region of approximately 350 bp containing a modified TATAA motif and several GC boxes. The chromosomal location of the mouse gene was determined by SSCP and fluorescence in situ hybridization and found to be at chromosome 10, band 4, between markers D1OMit5 -2.3 +/- 1.6 cM -col13a1 - 3.4+/-1.9 cM - D1OMit15. This result indicates that the mouse type XIII collagen gene and its human counterpart are located in chromosomal segments with conserved syntenies (The GenBank accession numbers for the mouse gene are AF063666-AF063693. The new GenBank accession number for the 5' end of the human type XIII collagen gene is AF071009).
Subject(s)
Collagen/genetics , Exons , Introns , Animals , Base Sequence , Binding Sites , Chromosome Mapping , DNA, Complementary , Electrophoresis, Gel, Pulsed-Field , Humans , Mice , Molecular Sequence Data , Promoter Regions, Genetic , Restriction Mapping , Transcription, GeneticABSTRACT
In this longitudinal study, we investigated the relationship of birth order and the age of mother and father to the gender of 1795 newborns (mean +/- SD 12.5 +/- 1.6 per mother) of 143 grand grand multiparous (i.e women who have had >10 deliveries). The frequency of boys was 52.2% in the group of 1st to 9th paras and 46.2% in the group of 10th to 20th paras (P = 0.022). Mothers aged > or =35 years had 7.0% more female than male newborns (P = 0.024). The respective figure for fathers was 5.6% (P = 0.023). The interpregnancy interval evaluated for 96 mothers with 1091 deliveries had no correlation with the gender of the infants. In the stepwise logistic regression analysis, the age of the mothers remained the only significant independent factor for the shift from a male to a female majority in the newborns (P = 0.0389). The present data thus indicate that the age of the mother is the factor which explains why grand grand multiparous women deliver more girls than boys.
Subject(s)
Sex Ratio , Adolescent , Adult , Aging , Female , Humans , Infant, Newborn , Logistic Models , Male , Middle Aged , Parity , PregnancyABSTRACT
In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.
Subject(s)
Enoyl-CoA Hydratase/chemistry , Liver/ultrastructure , Microbodies/enzymology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA, Complementary/chemistry , Enoyl-CoA Hydratase/metabolism , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Polymerase Chain Reaction , Rats , Sequence AlignmentABSTRACT
The genes for type XIII collagen (COL13A1) and prolyl 4-hydroxylase (P4HA) were previously assigned to human chromosome 10q by radioactive in situ hybridization. Here we have applied fluorescence in situ hybridization combined with targets representing different levels of resolution to determine, first, the order of these genes along chromosome 10; second, their transcriptional orientation; and third, the distance between these genes. The order along the chromosome was determined to be centromere-COL13A1-P4HA-telomere using mechanically stretched chromosomes. By combining the data from stretched chromosomes and interphase nuclei, we found that the transcriptional orientation were tail to tail (COL13A1 3'-3' P4HA). The distance between these genes was measured by fiber FISH to be approximately 550 kb.
Subject(s)
Chromosomes, Human, Pair 10 , In Situ Hybridization, Fluorescence/methods , Procollagen-Proline Dioxygenase/genetics , Transcription, Genetic , Centromere/genetics , Humans , Telomere/geneticsABSTRACT
Over 11,000 bp of previously undefined sequences of the human COL2A1 gene were defined. The results made it possible to compare the intron structures of a highly complex gene from man and mouse. Surprisingly, the sizes of the 53 introns of the two genes were highly conserved with a mean difference of 13%. After alignment of the sequences, 69% of the intron sequences were identical. The introns contained consensus sequences for the binding of over 100 different transcription factors that were conserved in the introns of the two genes. The first intron of the gene contained 80 conserved consensus sequences and the remaining 52 introns of the gene contained 106 conserved sequences for the binding of transcription factors. The 5'-end of intron 2 in both genes had a potential for forming a stem loop in RNA transcripts.