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F Ramirez

Publications and source records attributed to F Ramirez.

At least 55 records · Page 3Linked to original sources
PubMed1990

Pro-alpha 1(XI) collagen. Structure of the amino-terminal propeptide and expression of the gene in tumor cell lines.

We have determined the nucleotide sequence of several overlapping cDNA clones encoding the amino-terminal portion of human alpha 1(XI) procollagen. These experiments have revealed that this domain of the pro-alpha(XI) chain displays structural features common to other fibrillar procollagen molecules, such as a putative amino-terminal proteinase cleavage site and an interrupted collagenous segment. In the latter, structural similarities were noted when alpha 1(XI) was compared with alpha 1(II) and alpha 2(V) procollagens. Overall, however, the amino-terminal region of pro-alpha 1(XI) differs greatly in composition and size from that of other fibrillar chains. Nearly three-fourths of this domain is in fact composed of a 383-amino acid globular region in which a 3-cysteine cluster signals the transition to a long and highly acidic carboxyl-terminal segment. Finally, the unrestricted expression of this cartilage-specific collagen gene has been confirmed by the finding of high levels of pro-alpha 1(XI) mRNA in two human rhabdomyosarcoma cell lines.

Amino Acid Sequence↗
PubMed1990

Complex and diversified regulatory programs control the expression of vertebrate collagen genes.

The collagens represent a family of structurally related but genetically distinct proteins whose function is essential to maintaining the integrity of vertebrate organs. In addition to their supportive roles, collagens influence a variety of developmental programs and physiological processes. Transcription of collagen genes is controlled by a series of complex interactions between cis-acting regulatory elements and trans-acting nuclear factors that have positive or negative effects on gene expression. Collagen synthesis relies on the timely utilization of diversified regulatory programs that employ tissue and cell-type specific promoters and enhancers. Some of these programs lead to the production of structurally variant chains in different tissues, while others shut down synthesis of a specific collagen type during cell differentiation. Still others control collagen expression in distinct cell lineages. The number, complexity, and variety of the mechanisms leading to the diversified expression of the collagen genes illustrate the unique contribution of this family of proteins to multicellular organogenesis.

Animals↗
PubMed1990

An intron mutation in the human alpha 1(I) collagen gene alters the efficiency of pre-mRNA splicing and is associated with osteogenesis imperfecta type II.

This study describes a homozygous, G----A transition at the moderately conserved +5 position within the splice donor site of intron 14 in the human alpha 1(I) collagen gene. The mutation reduced the efficiency of normal splice-site selection since the exon upstream of the mutation was spliced alternatively. Moreover, the extent of alternative splicing was sensitive to the temperature at which the mutant cells were grown, suggesting that the mutation directly affected spliceosome assembly. To achieve exon skipping, this effect must be propagated so as to disrupt the selection of a second splice site in the adjacent intron.

Adenine↗
PubMed1990

Tumor necrosis factor-alpha and interferon-gamma suppress the activation of human type I collagen gene expression by transforming growth factor-beta 1. Evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels.

Regulation of human type I procollagen gene expression was studied in cultured fibroblasts both at the transcriptional and posttranscriptional level. Transcriptional regulation was examined in cultures transfected with a human pro alpha 2(I) collagen promoter/reporter gene (chloramphenicol acetyltransferase) construct, while posttranscriptional regulation was assessed by parallel determinations of type I procollagen mRNA steady-state levels. Transforming growth factor-beta 1 (TGF-beta 1) elicited a marked, approximately 5-23-fold, enhancement of pro alpha 2(I) collagen promoter activity, which was accompanied by an elevation of type I procollagen mRNA levels. This enhancement of gene expression was suppressed by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), as determined at mRNA steady-state level, but two distinct mechanisms were involved. TNF-alpha suppressed the pro alpha 2(I) collagen promoter activity, whereas IFN-gamma had only a minimal effect at transcriptional level. The effects of TNF-alpha and IFN-gamma were synergistic, suggesting that combination of these two factors may potentially provide pharmacologic means to counteract tissue deposition of collagen in diseases involving TGF-beta.

Animals↗
PubMed1989

IL-2 protects T cell hybrids from the cytolytic effect of glucocorticoids. Synergistic effect of IL-2 and dexamethasone in the induction of high-affinity IL-2 receptors.

IL-2-independent CD8+ rat x BW5147 T cell hybridomas are highly sensitive to treatment with 10(-6) M dexamethasone. This glucocorticoid analog induces a rapid DNA fragmentation with a pattern similar to that observed during glucocorticoid-induced killing of mouse thymocytes, which suggests the activation of a similar specific endonuclease. Among these hybrids, we select variants expressing low affinity IL-2R, as measured by IL-2 binding assay and by the cell surface expression of the IL-2Rp55 Ag (rat CD25 recognized by OX-39 mAb). These OX-39+ IL-2 independent hybrids (named V type) are protected from the toxic action of dexamethasone by IL-2. The addition of IL-2 to V type cells induces the expression of a low number of high affinity IL-2R, which is strongly potentiated by the simultaneous addition of dexamethasone. Furthermore, dexamethasone is strongly synergistic with IL-2 in the induction of mRNA p55/IL-2R, which could be observed 6 h after the treatment. These data suggest that the utilization of the IL-2-R signaling pathway may induce an effective protection against glucocorticoid toxicity in mature T cells. Finally, we proved that the upregulation of IL-2R by IL-2 is strongly potentiated by glucocorticoids, which implies a new role for these agents in the immune system.

Animals↗
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↗