SEARCH · PubMed Health

Results for “Codon, Initiator”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,045 records · Page 58Linked to original sources

PubMed1991

GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae.

The GCD2 protein is a translational repressor of GCN4, the transcriptional activator of multiple amino acid biosynthetic genes in Saccharomyces cerevisiae. We present evidence that GCD2 has a general function in the initiation of protein synthesis in addition to its gene-specific role in translational control of GCN4 expression. Two temperature-sensitive lethal gcd2 mutations result in sensitivity to inhibitors of protein synthesis at the permissive temperature, and the gcd2-503 mutation leads to reduced incorporation of labeled leucine into total protein following a shift to the restrictive temperature of 36 degrees C. The gcd2-503 mutation also results in polysome runoff, accumulation of inactive 80S ribosomal couples, and accumulation of at least one of the subunits of the general translation initiation factor 2 (eIF-2 alpha) in 43S-48S particles following a shift to the restrictive temperature. The gcd2-502 mutation causes accumulation of 40S subunits in polysomes, known as halfmers, that are indicative of reduced 40S-60S subunit joining at the initiation codon. These phenotypes suggest that GCD2 functions in the translation initiation pathway at a step following the binding of eIF-2.GTP.Met-tRNA(iMet) to 40S ribosomal subunits. consistent with this hypothesis, we found that inhibiting 40S-60S subunit joining by deleting one copy (RPL16B) of the duplicated gene encoding the 60S ribosomal protein L16 qualitatively mimics the phenotype of gcd2 mutations in causing derepression of GCN4 expression under nonstarvation conditions. However, deletion of RPL16B also prevents efficient derepression of GCN4 under starvation conditions, indicating that lowering the concentration of 60S subunits and reducing GCD2 function affect translation initiation at GCN4 in different ways. This distinction is in accord with a recently proposed model for GCN4 translational control in which ribosomal reinitiation at short upstream open reading frames in the leader of GCN4 mRNA is suppressed under amino acid starvation conditions to allow for increased reinitiation at the GCN4 start codon.

Amino Acids↗

PubMed1984

Nucleotide sequence of the temperate Bacillus subtilis bacteriophage SPO2 DNA polymerase gene L.

Temperate Bacillus subtilis phage SPO2 codes for a phage-specific DNA polymerase. The polymerase gene has been cloned, and its nucleotide sequence has been determined. Within the sequence there is an open reading frame starting with a TTG and ending with three consecutive translational stop codons. Ten base pairs upstream from the proposed TTG initiation codon there is a probable ribosome-binding site with a calculated free energy of interaction with the 3' end of B. subtilis 16S rRNA of -15 kcal (-63 kJ)/mol. Based on the sequence and the expression of the polymerase gene in three different hybrid plasmids, we conclude that this open reading frame is the structural gene for SPO2 DNA polymerase. The predicted molecular weight of the polymerase is 72,486. In hybrid plasmid pJB74, the terminal triplet of an open reading frame with coding capacity for a protein of ca. 10 kilodaltons overlaps with the translational initiation triplet TTG of the polymerase gene. We speculate that transcription and translation of this open reading frame can influence the amount of phage DNA polymerase made in SPO2-infected bacteria.

Bacillus subtilis↗

PubMed1990

The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum contains three promoters specific for growth, aggregation, and late development.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) plays essential roles throughout the development of Dictyostelium discoideum. It is crucial to cellular aggregation and to postaggregation morphogenesis. The phosphodiesterase gene is transcribed into three mRNAs, containing the same coding sequence connected to different 5' untranslated sequences, that accumulate at different times during the life cycle. A 1.9-kilobase (kb) mRNA is specific for growth, a 2.4-kb mRNA is specific for aggregation, and a 2.2-kb mRNA is specific for late development and is only expressed in prestalk cells. Hybridization of RNA isolated from cells at various stages of development with different upstream regions of the gene indicated separate promoters for each of the three mRNAs. The existence of specific promoters was confirmed by fusing the three putative promoter regions to the chloramphenicol acetyltransferase reporter gene, and the analysis of transformants containing these constructs. The three promoters are scattered within a 4.1-kilobase pair (kbp) region upstream of the initiation codon. The late promoter is proximal to the coding sequence, the growth-specific promoter has an initiation site that is 1.9 kbp upstream of the ATG codon, and the aggregation-specific promoter has an initiation site 3 kbp upstream.

Amino Acid Sequence↗

PubMed1982

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

Amino Acid Sequence↗

PubMed1992

The psbL gene from bell pepper (Capsicum annuum): plastid RNA editing also occurs in non-photosynthetic chromoplasts.

We have determined the nucleotide sequence of the plastid psbL gene from bell pepper. This gene has an ACG as a first codon. Isolation of RNA from pepper leaves and ripe fruits and subsequent sequencing of the psbL cDNA revealed that this ACG codon is post-transcriptionally edited into an AUG initiation codon in both leaves and fruits. These data indicate that the RNA editing machinery which exists in chloroplasts is still functional in chromoplasts from ripe fruits.

Amino Acid Sequence↗

PubMed1999

The nonrandom location of synonymous codons suggests that reading frame-independent forces have patterned codon preferences.

Biased codon usage is common in eukaryotic and prokaryotic genes. Evidence from Escherichia, Saccharomyces, and Drosophila indicates that it favors translational efficiency and accuracy. However, to date no functional advantages have been identified in the codon-anticodon interactions involving the most frequently used (preferred) codons. Here we present evidence that forces not related to the individual codon-anticodon interaction may be involved in determining which synonymous codons are preferred or avoided. We show that the "off-frame" trinucleotide motif preferences inferrable from Drosophila coding regions are often in the same direction as Drosophila's "in-frame" codon preferences, i.e., its codon usage. The off-frame preferences were inferred from the nonrandomness of the location of confamilial synonymous codons along coding regions-a pattern often described as a context dependence of nucleotide choice at synonymous positions or as codon-pair bias. We relied on randomizations of the location of confamilial codons that do not alter, and cannot be influenced by, the encoded amino acid sequences, codon usage, or base composition of the genes examined. The statistically significant congruency of in-frame and off-frame trinucleotide preferences suggests that the same kind of reading-frame-independent force(s) may also influence synonymous codon choice. These forces may have produced biases in codon usage that then led to the evolution of the translational advantages of these motifs as preferred codons. Under this scenario, tRNA pool size differences between preferred and nonpreferred codons initially were evolved to track the default overrepresentation of codons with preferred motifs. The motif preference hypothesis can explain the structuring of codon preferences and the similarities in the codon usages of distantly related organisms.

Amino Acid Sequence↗

PubMed1989

Identification of the 5' end of the rubella virus subgenomic RNA.

The 5' end of the subgenomic RNA of rubella virus was determined by primer extension. The Maxam-Gilbert sequence ladder of the primer extension product contained a determinable sequence which was colinear with the complement of the sequence of the genomic RNA through nucleotide 3325 from the 3' end of the genomic RNA and a pair of bands in every lane above the determinable sequence. These results indicated that synthesis of the subgenomic RNA is initiated internally on the minus-polarity genome-length RNA template and that the length of subgenomic RNA is 3327 nucleotides excluding the poly(A) tail. There are thus 77 nucleotides between the 5' end of the subgenomic RNA and the first AUG, which is the initiation codon for the structural protein open reading frame. The initiation site of rubella virus subgenomic RNA synthesis is 20 nucleotides downstream from a block of 28 nucleotides which shares homology with the nucleotide sequence which is conserved at the subgenomic RNA initiation site in the Alphaviruses, the other genus of Togaviruses.

Animals↗

PubMed1994

Organization of the bovine gene encoding the endothelial nitric oxide synthase.

The bovine endothelial nitric oxide synthase gene plus 2.9 kilobases of 5'-flanking sequence has been isolated and characterized. The gene spans 20 kilobases and contains 26 exons and 25 introns. Two transcription start sites have been determined by primer extension analysis which are located 170 and 240 base pairs upstream, respectively, from the methionine translational initiation codon. Evidence supporting the upstream boundary region for transcriptional initiation was also obtained by reverse transcription-polymerase chain reaction. The 5'-flanking region lacks a typical TATA box but contains numerous putative transcription factor binding sites. These include consensus sequences for an AP-1 site, an NF-1 site, a tumor necrosis factor responsive element, two sterol regulatory elements, 3 acute-phase response element, two sterol regulatory elements, 3 acute-phase response elements, 6 GATA motifs, 16 CACCC boxes, 5 Sp1 sites, 15 estrogen half-palindromic motifs, and 9 fluid shear stress-responsive elements. The isolated gene promoter directs basal transcription of a luciferase reporter gene when transiently transfected into bovine aortic endothelial cells. High sequence homology of the promoter region to the human endothelial nitric oxide synthase gene promoter (75% nucleotide identity in 1.6 kilobases of 5'-flanking sequence) suggests evolutionary conservation of transcriptional regulation. Isolation and characterization of the bovine endothelial nitric oxide synthase gene should facilitate further investigation of mechanisms by which gene expression is regulated.

Amino Acid Oxidoreductases↗

PubMed2000

Alternative translation initiation of human fibroblast growth factor 2 mRNA controlled by its 3'-untranslated region involves a Poly(A) switch and a translational enhancer.

Five fibroblast growth factor 2 (FGF-2) isoforms are synthesized from human FGF-2 mRNA by a process of alternative initiation of translation. The regulation of FGF-2 isoform expression by the mRNA 5823-nucleotide-long 3'-untranslated region containing eight alternative polyadenylation sites was examined. Because previous studies had shown that FGF-2 expression was regulated in primary cells but not in transformed cells, primary human skin fibroblasts were used in this study. Using an approach of cell transfection with synthetic reporter mRNAs, a novel translational enhancer (3'-TE) was identified in the 1370-nucleotide mRNA segment located upstream from the eighth poly(A) site. Deletion mutagenesis showed that the 3'-TE was composed of two domains with additive effects. The 3'-TE exhibited the unique feature of modulating the use of FGF-2 alternative initiation codons, which favored the relative expression of CUG-initiated isoforms. Interestingly, the use of an alternative polydenylation site removing the 3'-TE was detected in skin fibroblasts in response to heat shock and cell density variations. At high cell densities, 3'-TE removal was correlated with a loss of CUG-initiated FGF-2 expression. These data show that the FGF-2 mRNA 3'-untranslated region is able to modulate FGF-2 isoform expression by the coupled processes of translation activation and alternative polyadenylation.

3' Untranslated Regions↗

PubMed1997

Beta-thalassaemia in indigenous Belgians: an update.

Beta-thalassaemia, a widespread autosomal recessive disorder, occurs sporadically in Northern and Western European countries. Molecular analysis of the beta-globin gene has been carried out in 30 members of 15 unrelated indigenous Belgian families which presented with non sideropenic hypochromic and microcytic anaemia. For all of them, extensive search failed to find an ancestor at risk for the disease. The beta-globin genes were first screened for frequent beta-thalassemic mutations by dot-blot hybridization with specific radiolabeled oligonucleotide probes. Direct automated fluorescence-based DNA sequencing and, in one case, Southern blotting were also used. All the 30 patients were found to be heterozygous for a beta-thalassemic mutation. Eight different mutations were identified. Among these, four are commonly found in the Mediterraneans: codon 8 (-AA), IVS-I-1 (G-->A), IVS-1-6 (T-->C) and codon 39 (C-->T); three have occasionally been reported: initiation codon (T-->C) and codon 35 (C-->A) and a rare deletion of 12.6 kb which removes all the beta-globin gene and its flanking regions. A new mutation, a -CC deletion at codon 38/39 was found in one family. These results both at the biological and molecular level show that beta-thalassaemia exist in indigenous Belgian families with no known ancestor a risk for the disease. They also show that clinicians and biologists should keep in mind the existence of beta-thalassaemia in indigenous Belgian families when investigating hypochromic and microcytic anaemia in patients whom the past familial history does not evocate a risk for the disease.

Adenine↗

PubMed1985

Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins.

The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC. By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype. In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift. The identity of the envZ gene product was confirmed by immunoprecipitation. M13 dideoxy sequencing of the DNA around the wild-type ompR-envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented. A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence. The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products.

Amino Acid Sequence↗

PubMed1994

Cloning and sequence analysis of the rpsL and rpsG genes of Mycobacterium smegmatis and characterization of mutations causing resistance to streptomycin.

The Mycobacterium smegmatis rpsL and rpsG genes, encoding the ribosomal proteins S12 and S7, were cloned, and their DNA sequence was determined. The third nucleotide of the S12 termination codon overlapped the first nucleotide of the S7 translation initiation codon. A collection of 28 spontaneous streptomycin-resistant mutants of M. smegmatis were isolated. All had single-base-pair substitutions in the rpsL gene which were changed to a streptomycin-sensitive phenotype by complementation with a low-copy-number plasmid carrying the wild-type M. smegmatis rpsL gene. A total of eight different mutations were found in two specific regions of the rpsL gene. Fifty-seven percent (16 of 28) altered the Lys codon at position 43. Forty-six percent of the mutations (13 of 28) were due to a transition changing an AAG Lys codon to an AGG Arg codon, with eight changes at codon 43 and five at codon 88.

Amino Acid Sequence↗

PubMed2005

SAT: a late NS protein of porcine parvovirus.

The genomes of all members of the Parvovirus genus were found to contain a small open reading frame (ORF), designated SAT, with a start codon four or seven nucleotides downstream of the VP2 initiation codon. Green fluorescent protein or FLAG fusion constructs of SAT demonstrated that these ORFs were expressed. Although the SAT proteins of the different parvoviruses are not particularly conserved, they were all predicted to contain a membrane-spanning helix, and mutations in this hydrophobic stretch affected the localization of the SAT protein. SAT colocalized with calreticulin in the membranes of the endoplasmic reticulum and the nucleus. A knockout mutant (SAT(-)), with an unmodified VP sequence, showed a "slow-spreading" phenotype. These knockout mutants could be complemented with VP2(-) SAT(+) mutant. The SAT protein is a late nonstructural (NS) protein, in contrast to previously identified NS proteins, since it is expressed from the same mRNA as VP2.

Amino Acid Sequence↗

PubMed1990

The production, purification, and bioactivity of recombinant bovine trophoblast protein-1 (bovine trophoblast interferon).

Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon- alpha that has a role in maternal recognition of pregnancy in cattle. Here we describe production of bTP-1 by recombinant procedures in Escherichia coli. A bTP-1 gene was constructed which lacked the codons representing the signal sequence and provided a Met initiation codon ahead of the TGT codon encoding Cys1 of the mature protein. This construct was placed under the control of the Trp promoter within the expression vector pTrp2. Expression occurred optimally in E. coli D112 in the absence of tryptophan and in the presence of 0.5% acid-hydrolyzed casein (casamino acids) when 0.5 mM indole acetic acid was included in the medium. The bTP-1 was deposited in inclusion bodies and accounted for as much as 27% of the total cellular protein. The inclusion bodies were isolated by differential centrifugation and washed. The bTP-1 was solubilized by use of guanidinium-HCI and 2-mercaptoethanol and allowed to renature in air. Final purification was achieved by anion exchange chromatography on DEAE-cellulose. The yield of purified product, which had an antiviral activity greater than 10(8) international reference units/mg, was approximately 20 mg/liter. The recombinant bTP-1 was relatively stable to freeze-thawing and frozen storage, and could induce the production of an acidic protein of 70,000 mol wt in cultured explants of endometrium prepared from ewes on day 13 of the estrous cycle. The latter protein is a characteristic product of interferon-alpha action on uterine tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Structural organization of nuclear gene for subunit Vb of mouse mitochondrial cytochrome c oxidase.

We have reported recently the isolation of a cDNA for nuclear encoded subunit Vb of mouse cytochrome c oxidase by screening mouse bone marrow and kidney cDNA libraries. In the present study, this cDNA was used as a probe to screen a mouse genomic library and isolate the complete gene encoding subunit Vb. Southern blot hybridization of mouse genomic DNA with the cDNA probe suggested the occurrence of multiple genes including many retroinserts. Restriction analysis followed by Southern blot hybridization of genomic clones was used to identify the putative retroinserts from the intron containing genes. Of the 10 initial genomic clones isolated, one clone (MG3) showing the most complex hybridization pattern was found to contain the complete gene for subunit Vb. The DNA sequence analysis show that the subunit Vb gene contains four exons of 149, 73, 99, and 189 bases interrupted by three relatively small introns of 520, 165, and 648 nucleotides in a gene spanning about 2.5 kilobase pairs. As determined by a combination of primer extension and S1 protection analyses, the major transcription start site appears to be located 49 nucleotides upstream of the translation initiation codon. The ability of the 5' upstream DNA to initiate transcription was studied using the chloramphenicol acetyltransferase (CAT) expression plasmids in NIH 3T3 cells. Using this system we observed that a segment of the gene spanning nucleotides -574 to +45 can drive the transcription of CAT gene in an orientation dependent manner. The upstream region of subunit Vb gene lacks the TATA and CAAT elements, although it contains several GC rich elements and a pyrimidine rich stretch around the transcription start site.

Amino Acid Sequence↗

PubMed1986

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase.

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.

Amino Acid Sequence↗

PubMed1994

Cloning, sequence analysis, and expression of the genes encoding the two subunits of the methylotrophic bacterium W3A1 electron transfer flavoprotein.

The genes encoding the two different subunits of the electron transfer flavoprotein (ETF) from the methylotrophic bacterium W3A1 have been identified, cloned, and sequenced. A 0.8-kilobase pair DNA fragment was generated for use as a molecular probe by the amplification of genomic sequences using the polymerase chain reaction and a primer pair with degenerate sequences derived from the NH2-terminal amino acid sequences determined for the ETF subunits purified from W3A1. The screening of a partial genomic minilibrary containing size-selected BamHI-SalI fragments using this probe identified a 2.2-kilobase pair insert containing the complete coding sequences for both W3A1 ETF subunits. The genes are arranged in tandem in the genomic DNA with only 2 bases between the TAG translation termination codon of the small subunit and the ATG translation initiation codon of the large subunit. The deduced amino acid sequences of each of the W3A1 ETF subunits exhibit only approximately 30% identity with the corresponding subunits of the ETF from human, rat, and Paracoccus denitrificans, which as a group are greater than 50% identical. Thus, the ETF from W3A1 may exhibit some unique structural features that, like other differences in some of its physical and functional properties, may distinguish this ETF from others in this family. A highly homologous region near the COOH terminus of the large subunit in all the ETF proteins was found to contain a sequence that matches in several ways the ADP-binding motif of flavoproteins and other dinucleotide-binding proteins, suggesting that the large subunit forms a portion of the FAD (or AMP) binding site in these proteins. Under control of the tac promoter, the cloned ETF subunit genes were co-expressed in Escherichia coli producing the heterodimeric holoprotein with physical, spectral, and electron-accepting properties essentially identical to the ETF isolated from W3A1. The recombinant ETF serves as the electron acceptor for W3A1 trimethylamine dehydrogenase in vitro, accumulating as the air-stable anionic semiquinone in the presence of excess trimethylamine. Fully reduced ETF could not be obtained even after prolonged enzymatic reduction.

Amino Acid Sequence↗

PubMed1996

Posttranslational regulation of type I collagen in corneal endothelial cells.

PURPOSE: Type I collagen synthesis in corneal endothelial cells does not correlate with steady state collagen RNA levels; although substantial amounts of alpha 2(I) collagen RNA are present in these cells, type I collagen is not detected. This allowed the authors to investigate the possibility of posttranscriptional control of type I collagen in corneal endothelial cells. METHODS: The alpha 2(I) collagen RNA structures of normal and modulated corneal endothelial cells were analyzed by S1 nuclease protection analysis, whereas the nucleotide sequences were obtained by rapid amplification of cDNA ends technique. In situ hybridization of type I collagen was demonstrated with immunofluorescence; synthesis and degradation of the molecule were analyzed by pulse-chase experiments and then by immunoprecipitation with antiprocollagen I antibody. RESULTS: The cDNA covering the 5'-untranslated region (UTR) of alpha 2(I) collagen RNA obtained from normal corneal endothelial cells and from modulated corneal endothelial cells that predominantly produce type I collagen demonstrate identical sequences in their 5' untranslated and coding sequences. In both mRNA, the length of the 5'-untranslated segment is 127 nucleotides. There were also two AUG codons; the second AUG codon, which is 17 nucleotides upstream from the translation initiation codon, is conserved, as observed in human and chicken alpha 2(I) mRNA. When the sequence covering the 3'-UTR of corneal endothelial alpha 2(I) mRNA was compared with that of alpha 2(I) mRNA obtained from the modulated cells, there were differences in only two nucleotides. The length of the 3'-untranslated segment of each mRNA is 297 nucleotides up to the consensus polyadenylation recognition site (AAUAAAAUAAA), which both cells use. Immunofluorescent staining of corneal tissue in vivo demonstrated that the corneal endothelium stains with anti-type I collagen antibodies, but there is no staining in the underlying Descemet's membrane. In pulse-chase experiments, the newly synthesized type I procollagen, composed of pro alpha 1(I) and pro alpha 2(I) chains as determined by V8 protease peptide mapping, reached the highest intracellular level at 45 minutes, after which its detection decreased. Cells chased for 120 minutes demonstrated no trace of type I procollagen in the cell layer; medium fractions showed no detectable type I procollagen during the entire 120-minute chase. CONCLUSIONS: These results suggest that type I collagen is synthesized in corneal endothelial cells and that such undesired expression is regulated at the posttranslational level, perhaps by intracellular degradation.

Amino Acid Sequence↗