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F Ramirez

Publications and source records attributed to F Ramirez.

At least 73 records · Page 4Linked to original sources
PubMed1989

Glycine to serine substitution in the triple helical domain of pro-alpha 1 (II) collagen results in a lethal perinatal form of short-limbed dwarfism.

Previous biochemical studies on cartilage tissue from a proband with Type II achondrogenesis-hypochondrogenesis (Godfrey, M., and Hollister, D. W. (1988) Am. J. Hum. Genet. 43, 904-913) indicated heterozygosity for a structural abnormality in the triple helical domain of pro-alpha 1 (II) collagen. Here we demonstrate that the mutation in the type II procollagen gene is a single base change that converts the codon for glycine (GGC) at amino acid 943 of the alpha 1 (II) chain to a codon for serine (AGC). The substitution disrupts the invariant Gly-X-Y structural motif necessary for perfect triple helix formation and leads to extensive overmodification, intracellular retention, and reduced secretion of type II collagen. These findings confirm the proposal that new dominant mutations in the type II procollagen gene may account for some cases of Type II achondrogenesis-hypochondrogenesis. Since recent studies (Lee, B., Vissing, H., Ramirez, F., Rogers, D., and Rimoin, D. (1989) Science 244, 978-980) have identified a dominantly inherited type II procollagen gene deletion in a non-lethal form of skeletal dysplasia, namely spondyloepiphyseal dysplasia, the data more generally demonstrate that different type II procollagen gene mutations eventuate in a wide and diverse spectrum of clinical phenotypes.

Amino Acid Sequence↗
PubMed1989

Temperature-dependent expression of a collagen splicing defect in the fibroblasts of a patient with Ehlers-Danlos syndrome type VII.

In this article we report the characterization of the molecular lesion in a patient with Ehlers-Danlos syndrome Type VII and provide evidence that a de novo substitution of the last nucleotide of exon 6 in one allele of the pro-alpha 2(I) collagen gene produces normally spliced mRNA and transcripts from which exon 6 sequences have been outspliced as well. Unexpectedly, the expression of the alternative splicing was found to be temperature-dependent, for missplicing in cellula is effectively abolished at 31 degrees C and gradually increases to 100% at 39 degrees C. In contrast, in a similar patient harboring a substitution in the obligatory GT dinucleotide of the 5' splice site of intron 6, complete outsplicing of exon 6 sequences was found at all temperatures.

Amino Acid Sequence↗
PubMed1989

Cloning and analysis of the 5' portion of the human type-III procollagen gene (COL3A1).

We have isolated overlapping clones containing the 5'-terminal portion of the human pro-alpha 1(III) collagen gene (COL3A1). This has enabled us to extend our previous studies and thus generate a restriction map of nearly 64 kb of DNA encompassing all of COL3A1 and more than 20 kb of flanking sequences. Aside from the complete nucleotide and amino acid sequences of type-III N-pre-propeptide, this study has established the number of the corresponding exons, whose relative organization deviates from the pattern observed in the analogous regions of type-I procollagen genes, COL1A1 and COL1A2. Moreover, we have sequenced 1628 bp of the 5'-flanking region of COL3A1, from the transcription start point (tsp) to an AluI repetitive element. Pairwise comparison with the analogous segment of the mouse gene has showed 78% sequence similarity in nearly 270 bp immediately preceding the tsp and including the TATA element and a presumptive NF-1 binding site. Relatively close to the tsp, but upstream from the region of homology with the murine gene, a potential AP-1 binding site has also been identified.

Amino Acid Sequence↗
PubMed1989

Identification of the molecular defect in a family with spondyloepiphyseal dysplasia.

Spondyloepiphyseal dysplasias (SED) are a heterogeneous group of inherited disorders characterized by disproportionate short stature and pleiotropic involvement of the skeletal and ocular systems. Evidence has suggested that SED may result from structural defects in type II collagen. To confirm the validity of this hypothesis, the structure of the "candidate" type II collagen gene (COL2A1) has been directly examined in a relatively large SED family. Coarse scanning of the gene by Southern blot hybridization identified an abnormal restriction pattern in one of the affected members of the kindred. Analysis of selected genomic fragments, amplified by the polymerase chain reaction, precisely localized the molecular defect and demonstrated that all affected family members carried the same heterozygous single-exon deletion. As a consequence of the mutation, nearly 90 percent of the assembled type II collagen homotrimers are expected to contain one or more procollagen subunits harboring an interstitial deletion of 36 amino acids in the triple helical domain.

Amino Acid Sequence↗
PubMed1989

Amino-terminal propeptide of human pro-alpha 2(V) collagen conforms to the structural criteria of a fibrillar procollagen molecule.

We have determined the nucleotide sequence of a cDNA clone encoding the amino-terminal portion of human alpha 2(V) procollagen and found that the structure of the 186-residue amino-terminal propeptide closely resembles those of the fibril-forming procollagens. Juxtaposed to a 26-residue leader peptide, pro-alpha 2(V) exhibits a characteristic cysteine-rich globular region followed by 24 Gly-X-Y repeats which are interrupted by two short non-collagenous sequences. Upon closer examination, each of these two sequences was noted to display structural motifs characteristic of either pro-alpha 1(I) and pro-alpha 1(III) collagens or pro-alpha 1(II) collagen, respectively. Finally, within the amino-terminal telopeptide, a putative amino-terminal proteinase cleavage site, Ala-Gln, was identified. This latter finding strongly suggests that the alpha 2(V) amino-terminal propeptide can be potentially processed and thus leaves unresolved the issue pertaining to the nature of the collagenase-resistant sequence that is retained by mature type V collagen molecules.

Amino Acid Sequence↗
PubMed1989

A base substitution in the exon of a collagen gene causes alternative splicing and generates a structurally abnormal polypeptide in a patient with Ehlers-Danlos syndrome type VII.

An unusual splicing mutation has been characterized in the pro alpha 1(I) collagen gene of a sporadic case of Ehlers-Danlos Syndrome Type VII. Cloning of primer extended cDNA in conjunction with R-looping experiments established that nearly half of the pro alpha 1(I) collagen gene transcripts are abnormally spliced, for they lack exon 6 sequences. Analysis of cloned genomic fragments revealed that one of the proband's alleles displays the substitution of an A for a G in the last nucleotide of exon 6. The change converts the normal Met (ATG) codon to Ile (ATA) and, in addition, obliterates a NcoI restriction site. The latter event was exploited to demonstrate the de novo nature of the mutation since DNA from the unaffected parents was fully digested with the enzyme, after in vitro amplification by the polymerase chain reaction. Further confirmation of the missplicing was obtained by transient expression into animal cells of allelic minigene constructs. Finally, Western blot analysis of cyanogen bromide cleaved collagen and nucleotide sequencing of appropriately selected cDNA clones demonstrated the production of relatively low amounts of correctly spliced molecules harboring the Ile substitution, as well.

Amino Acid Sequence↗
PubMed1989

Molecular defects of type III procollagen in Ehlers-Danlos syndrome type IV.

Fibroblasts from most patients with Ehlers-Danlos syndrome (EDS) type IV, a disorder characterized by fragility of skin, blood vessels, and internal organs, secrete reduced amounts of type III procollagen. In 7 of 8 cell strains analyzed, we found evidence of structural defects in half of the type III procollagen chains synthesized, such as deletions or bona fide amino acid substitutions, which cause delayed formation and destabilization of the collagen triple helix and, as a consequence, reduced secretion of the molecule. The data suggest that EDS type IV is often caused by heterozygosity for mutations at the COL3A1 locus, which affect the structure of type III procollagen. The triple-helical region of the molecule, like the homologous region of type I procollagen, appears to be particularly vulnerable.

DNA↗
PubMed1989

Organization of the exons coding for pro alpha 1(II) collagen N-propeptide confirms a distinct evolutionary history of this domain of the fibrillar collagen genes.

The organization of the exons coding for the N-terminal portion of human type II procollagen has been determined. Aside from inferring the previously unknown primary structure of type II N-propeptide, this study has revealed that this coding domain of the gene exhibits an organization uniquely distinct from those of type I and type III collagens. This finding substantiates the notion that the N-propeptide coding domains of the fibrillar collagen genes evolved under less stringent selection than those encoding the C-propeptide and triple helical regions.

Amino Acid Sequence↗
PubMed1989

Structure and developmental expression of a sea urchin fibrillar collagen gene.

We have isolated and characterized cDNA and genomic clones that specify a Paracentrotus lividus procollagen chain. The cDNAs code for 160 uninterrupted Gly-Xaa-Yaa triplets and a 252-amino acid carboxyl propeptide. Analysis of the deduced amino acid sequences indicated that the sea urchin polypeptide exhibits structural features that are characteristic of the fibril-forming class of collagen molecules. Partial characterization of two genomic recombinants revealed that the 3' end of the echinoid gene displays a complex organization that closely resembles that of a prototypical vertebrate fibrillar collagen gene. In situ and Northern (RNA) blot hybridizations established the size, time of appearance, and tissue distribution of the collagen transcripts in the developing sea urchin embryo. Collagen mRNA, approximately equal to 6 kilobases in size, is first detected in the forming primary mesenchyme cells of late blastulae where it progressively accumulates until the free swimming/feeding pluteus larval stage. Interestingly, collagen transcripts are also detected in the forming secondary mesenchyme cells of late gastrulae, and by the prism stage, their derivatives appear to be the most intensively labeled cells.

Amino Acid Sequence↗
PubMed1989

Pulmonary phaeohyphomycosis in a patient with hemoptysis.

A 79-year-old retired schoolteacher had a history of bronchiectasis. She developed recurrent hemoptysis requiring multiple blood transfusions. Exophiala dermatitidis was cultured repeatedly from bronchial lavages. To our knowledge, this is the first documented case of isolated pulmonary phaeohyphomycosis due to E dermatitidis, and it was successfully treated with amphotericin B and 5-fluocytosine.

Aged↗
PubMed1989

Molecular pathobiology of human collagens.

The fibril-forming collagens (types I-III, V and XI) represent a homogeneous and evolutionary related group of proteins and genes. In addition to serving as supportive elements, these macromolecules influence the spatial and ontogenic diversity of extracellular matrices, for they regulate a number of developmental programs and cellular activities, such as adhesion, proliferation and migration. Deranged expression of fibrillar collagen genes results in a number of inherited and acquired disorders which greatly affect the structural integrity of the organism. An understanding of collagen biosynthesis and regulation in normal and diseased states provides an opportunity to dissect biological problems which relate to a wide variety of subjects, including morphogenesis, relationships between structure and function of proteins, gene expression and human mutations.

Animals↗
PubMed1988

Cloning and sequencing of pro-alpha 1 (XI) collagen cDNA demonstrates that type XI belongs to the fibrillar class of collagens and reveals that the expression of the gene is not restricted to cartilagenous tissue.

We have isolated several overlapping cDNA clones encoding alpha 1(XI) collagen chains from human and rat cDNA libraries. Together the human cDNAs code for 335 uninterrupted Gly-X-Y triplets, and a 264-amino acid C-propeptide, while the rat cDNAs cover the entire C-propeptide and about a third of the triple-helical domain. Comparison of the human and rodent nucleotide sequences showed a 95% sequence similarity. The identification of the clones as alpha 1(XI) cDNAs was based on the complete identity between the amino acid sequences of three human alpha 1(XI) cyanogen bromide peptides and the cDNA-derived sequence. Examination of and the cDNA-derived amino acid sequence showed a variety of structural features characteristic of fibrillar-forming collagens. In addition, nucleotide sequence analysis of a selected portion of the corresponding human gene revealed the characteristic 54-base pair exon motif. We conclude therefore that pro-alpha 1 (XI) collagen belongs to the group of fibrillar collagen genes. We also suggest that the expression of this gene is not restricted to cartilage, as previously thought, since the cDNA libraries from which the clones were isolated, originated from both cartilagenous and noncartilaginous tissues.

Amino Acid Sequence↗
PubMed1988

Complete nucleotide sequence of the region encompassing the first twenty-five exons of the human pro alpha 1(I) collagen gene (COL1A1)

Dysfunctions of the genes coding for the two chains of the human type-I procollagen result in genetic disorders that affect the integrity of bone, ligaments, tendons, and other connective tissues. While the primary amino acid (aa) sequence of one of the two type-I subunits, pro alpha 2(I), has been derived in its entirety from the analysis of overlapping cDNAs, the sequence of the first 247 aa residues of the helical domain of the other polypeptide, pro alpha 1(I), had yet to be determined. To this end, we have sequenced nearly 4 kb of the human pro alpha 1(I) collagen gene and identified twelve open reading frames whose conceptual amino acid translation exhibits 95% homology to the first 247 aa of rat alpha 1(I) chain. Furthermore, with these and other data, some of which previously unpublished, we have derived the complete sequence of the first 7618 bp of the gene. This region comprises the 25 exons encoding the N-terminal pre-propeptide and five of the eight cyanogen-bromide-derived peptides. This information therefore represents a most useful reference for the characterization of molecular defects in individuals affected by various connective tissue disorders.

Amino Acid Sequence↗
PubMed1988

Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant.

Recent biochemical studies have shown that the fibroblasts from a patient with Ehlers-Danlos Syndrome Type VIIB produce nearly equal amounts of normal and shortened pro-alpha 2(I) collagen chains (Wirtz, M.K., Glanville, R. W., Steinmann, B., Rao, V. H., and Hollister, D. (1987) J. Biol. Chem. 262, 16376-16385). Compositional and sequencing studies of the abnormal pro-alpha 2(I) chain identified an interstitial deletion of 18 residues corresponding to the N-telopeptide of the collagen molecule. Since this region is encoded by a 54-base pair exon, number 6, the protein defect could have been caused by gene deletion, abnormal pre-mRNA splicing, or both. Here, in order to elucidate the molecular nature of this mutation we have analyzed the sequences of pro-alpha 2(I) collagen cDNA and genomic clones obtained from RNA and DNA of the patient's fibroblasts. Using oligomer-specific cloning we identified a cDNA that contains a 54-base pair deletion corresponding precisely to the sequence of exon 6. Identification of the normal gene was based on the finding of an identical sequence polymorphism in a normal cDNA and in the genomic clone derived from one of the two collagen alleles. The other gene, instead, displayed a base substitution (T to C) in the obligatory GT dinucleotide of the 5' splice-site sequence of intron 6. Analysis of nearly 100 base pairs immediately 5' to exons 5, 6, and 7, and 3' to exons 5 and 7 did not reveal any additional change. Therefore, the data strongly suggest that the observed GT-to-GC transition at the splice donor site of intron 6 generates an abnormally spliced mRNA in which the sequence of exon 5 is joined to the sequence of exon 7. Since skipping of exon 6 does not interfere with the coding frame of the mRNA, the resulting shortened polypeptide, albeit utilized in the assembly of a procollagen trimer, ultimately causes the Ehlers-Danlos Syndrome Type VII phenotype.

Alleles↗