Bovine lens 23, 21 and 19 kDa intrinsic membrane proteins have an identical amino-terminal amino acid sequence.

We have isolated bovine lens intrinsic membrane proteins (MP) having molecular masses of 19, 21 and 23 kDa. Limited amino acid sequence analysis of the amino-terminal portion of each of these polypeptides revealed a 100% homology in sequence for the number of residues determined (20 amino acids). Northern blot analysis of bovine lens mRNA using a labeled antisense oligonucleotide probe common to the amino acid sequence of these three peptides revealed a single band having an apparent molecular size of 0.8 kb. Taken together, these findings suggest a genetic commonality between these polypeptides.

Posttranscriptional regulation of the expression of CAD gene during differentiation of F9 teratocarcinoma cells by induction with retinoic acid and dibutyryl cyclic AMP.

We have studied the regulation of expression of the carbamoyl-phosphate synthetase II-aspartate transcarbamylase-dihydroorotase gene in F9 teratocarcinoma cells during their differentiation into parietal endoderm cells by induction with a combination of retinoic acid and dibutyryl cyclic AMP. Steady-state levels of CAD mRNA decreased by 7-fold in F9 cells following 120 h of retinoic acid and dibutyryl cyclic AMP induction as compared to levels in uninduced cells. Conversely, no apparent changes were found in the steady-state levels of beta-actin mRNA between induced and uninduced cells. Despite a 7-fold decrease in the steady-state levels of CAD mRNA, its rate of transcription remained the same between induced and uninduced cells, indicating a role for posttranscriptional mechanisms for its down regulation during retinoic acid- and dibutyryl cyclic AMP-induced differentiation of F9 cells. The cellular growth rate of F9 cells as determined by [3H]thymidine uptake and parallel cell counting decreased markedly during their induction with retinoic acid and dibutyryl cyclic AMP. Taken together, it is apparent that the expression of the CAD gene is cell-growth-dependent and its regulation in this system is at the posttranscriptional level.

Procollagen and collagen produced by normal bovine corneal stroma fibroblasts in cell culture.

Procollagen and collagen were isolated from the culture medium of normal bovine corneal stromal fibroblasts. DEAE-cellulose chromatography was used to separate the collagen molecules from the different procollagens present. One collagen and four procollagen peaks were isolated and biochemically characterized. All the procollagen fractions and the collagen fraction yielded, after limited pepsin or chymotrypsin digestion followed by CNBr digestion, molecules that correspond to (alpha 1)2 alpha 2 exclusively. Thus only type I collagen is found in the growth medium of of bovine corneal stromal fibroblast cultures. Each of the individual procollagen peaks contained pro-alpha chains having molecular weights of 120,000, 150,000, 165,000, 180,000, and 190,000 daltons, according to their elution position on DEAE-cellulose. The presence of type I procollagen molecules having pro-alpha chains of 165,000, 180,000, and 190,000 daltons has not previously been reported and probably represents higher-molecular-weight precursor intermediates. The amino acid compositions of the different procollagen fractions are unique, and each contains relatively large amounts of cysteine and tryptophan. Carbohydrate analysis, cyanogen bromide peptide analysis, electron microscopy of SLS-crystallities, and SDS-polyacrylamide gel electrophoresis were used to further characterize the procollagen and collagen molecules.

Gene mapping of human ocular connective tissue proteins. Assignment of the structural gene for corneal type I procollagen to human chromosome 7 in human corneal stroma-mouse fibroblast somatic cell hybrids.

Somatic cell hybrids between mouse and human corneal stroma fibroblasts have been used to identify the human chromosome responsible for the synthesis of human corneal type I procollagen. Twenty-six separate hybrid clones and subclones from three separate hybridization events were isolated in hypoxanthine-aminopterin-thymidine (HAT) selection medium and were used to assay for the production of human type I procollagen. Human and mouse chromosomes were identified in each hybrid clone by alkaline Giemsa staining of metaphase chromosomes spread and by isozyme analysis. We have found that human type I procollagen production segregates concordantly with human chromosome 7 and with beta-glucuronidase, another human chromosome 7 marker. The pro-alpha 1 gene and possibly the pro-alpha 2 gene appear to be encoded on human chromosome 7. Because we have previously assigned the gene for human skin type I procollagen to chromosome 17, our present data indicate that separate genes and control mechanisms must exist for skin and corneal type I procollagen.

Partial characterization of human collagen and procollagen secreted by human corneal stromal fibroblasts in cell culture.

Human corneal stromal fibroblasts were established into cell culture. Fractions highly enriched for collagen and procollagen were purified from the medium and cell layer. Analysis using polyacrylamide gel electrophoresis and DEAE cellulose chromatography indicated the presence of a heterogeneous population of procollagen molecules ranging in molecular weights from 200,000 to 120,000 daltons along with alpha1 and alpha2 collagen chains. Cyanogen bromide peptides of a corneal peptide fraction migrated concomitantly with type I standard cyanogen bromide peptides.

Biochemical characterization of procollagen-collagen synthesized by rabbit corneal endothelial cells in culture.

Collagen synthesis was studied in monolayer cultures of rabbit corneal endothelial cells by following [14C]proline and [3H]glucosamine or [3H]fucose incorporation into a fraction enriched for collagen and its precursor molecules. Sodium dodecyl sulfate gel electrophoresis of this fraction showed that it consisted of a high-molecular-weight (greater than 300,000 daltons) polypeptide. This component was collagenase sensitive and, in the presence of dithiothreitol, gave rise to two polypeptides of the apparent molecular weights of 200,000 and 160,000 daltons. Pepsin digestion of this material destroyed all the high-molecular-weight material and gave rise to a single collagenase-sensitive component of an apparent molecular weight of 115,000 daltons. This 115,000 dalton material is similar to previously observed basement membrane collagens, and the 160,000 and 200,000 dalton components are probably precursor chains of basement membrane collagen. The very-high-molecular-weight material (greater than 300,000 daltons) may represent a disulfide-linked complex of these precursor chains. DEAE-cellulose column chromatography confirmed the presence of a single procollagen species distinct from the collagen fraction. Amino acid analysis of collagen and procollagen fractions showed a decreased hydroxyproline value as compared with previously reported basement membrane collagens or collagen precursors.

The mouse lens fiber-cell intrinsic membrane protein MP19 gene (Lim2) and granule membrane protein GMP-17 gene (Nkg7): Isolation and sequence analysis of two neighboring genes.

PURPOSE: The lens fiber cell intrinsic membrane protein MP19 appears to play a key role in lens fiber cell structure or communication, and thus cataractogenesis. The goal of this study was to isolate and characterize the entire gene structure of the MP19 gene, termed Lim2, and to investigate gene sequences surrounding this lens-specific gene. METHODS: A 129/SvJ mouse genomic DNA library was screened using radioisotope labeled bovine MP19 cDNA. From this screening, an 11 kb genomic fragment was isolated which contained the entire Lim2 gene, and a neighboring gene, Nkg7, which codes for a 17 kDa granulocyte membrane protein termed GMP-17. The nucleotide sequence of this entire fragment was obtained using double strand automated sequencing techniques. Using CAT and green fluorescent protein reporter constructs, Lim2 5'-upstream promoter sequences were analyzed. RESULTS: An 11,182 base pair genomic clone containing the entire murine Lim2 gene and another downstream gene, Nkg7, was obtained and completely sequenced. These two genes are only 1,182 base pairs apart, from the poly(A) signal of the Lim2 gene to the published transcriptional start site of Nkg7. Interestingly, the protein coded for by Nkg7, GMP-17, is very similar to the product of the lens Lim2 gene, MP19, in many respects. Both proteins are transmembrane proteins, with each having 4 transmembrane loops. The amino acid sequence of the two proteins is 34% identical, and 49% with respect to similar amino acids. The size of mouse Lim2 is 5,896 base pairs from the transcriptional start site to the poly(A) signal, and contains five exons and four introns. Exons 2-5 of the Lim2 gene encode a polypeptide of 173 amino acids, having over 92% identity to human MP19. Using chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) reporter constructs, it was determined that about 160 bp of sequence upstream from the start of transcription is both necessary and sufficient for efficient expression levels as well as tissue specificity of expression. CONCLUSIONS: The mouse Lim2 gene is very similar to the human LIM2 gene, both having the same number of exons and introns. The coding nucleotide sequences from both species are 88% identical, and 92% identical at the amino acid level. In the immediate 5'-upstream region of these two genes, several highly conserved regions are observed. Due to the similarity of the MP19 and GMP-17 proteins, it is interesting to speculate that the lens MP19 and the lymphocyte-associated GMP-17 may have originated from one primordial gene which, through genetic drift, resulted in two separate proteins having similar functions in two widely separated tissue types.

Regional mapping of the human MP70 (Cx50; connexin 50) gene by fluorescence in situ hybridization to 1q21.1.

PURPOSE: Gap junctions play a critical role in the metabolic homeostasis and maintenance of transparency of fibers within the ocular lens. As part of a long-term effort to establish the relationship between lens gap junction proteins, normal lens development, and cataractogenesis, we report here the regional localization of the human MP70 (Connexin 50) gene. METHODS: Fluorescence in situ hybridization (FISH) was used to regionally map the human MP70 gene. The DNA probe contained the entire MP70 coding region within a clone isolated from a human genomic DNA library. RESULTS: The human gene encoding the lens intrinsic membrane protein MP70 was regionally mapped to q21.1 on the long arm of chromosome 1. CONCLUSIONS: This study confirms the previous provisional assignment of MP70 to human chromosome 1 and regionally localizes the gene to 1q21.1. When combined with previous mapping information, these data are consistent with the hypothesis that a genetic lesion in the gene encoding the lens intrinsic membrane protein MP70 may be the underlying molecular defect for zonular pulverulent (Coppock) cataract. Furthermore, these combined data support the hypothesis that other forms of human hereditary cataract may be the result of a mutation in one or more of the genes encoding gap junction proteins found in the ocular lens.

Identification of a mutation in the MP19 gene, Lim2, in the cataractous mouse mutant To3.

PURPOSE: Lim2, the gene encoding the second most abundant lens specific integral membrane protein, MP19, has recently been proposed as an ideal candidate gene for the cataractous mouse mutant, To3. The aim of this study was to screen the Lim2 gene in the To3 mutant for a genetic lesion that was correlated and consistent with the mutant phenotype. METHODS: Genomic DNA was isolated from both normal mouse parental strains as well as the heterozygous and homozygous To3 cataract mutant. PCR was used to generate overlapping fragments of the entire Lim2 gene from these DNAs. The coding regions, including splice junctions and the translational termination site, of these fragments were then sequenced. RESULTS: A single G -> T transversion was identified within the first coding exon of the Lim2 gene in the To3 mutant DNA. This DNA change results in the nonconservative substitution of a valine for the normally encoded glycine at amino acid 15 of the MP19 polypeptide. CONCLUSIONS: The identified genetic lesion in the Lim2 gene of the cataractous mouse mutant, To3, confirms Lim2 as an ideal candidate gene. Future transgenic experiments should provide proof or disproof of a causative relationship between the identified mutation and the cataractous phenotype. These studies indicate that MP19 may play an important role in both normal lens development and cataractogenesis, and warrants more intense investigation of its role within the ocular lens.

Lim2(To3) transgenic mice establish a causative relationship between the mutation identified in the lim2 gene and cataractogenesis in the To3 mouse mutant.

PURPOSE: Lim2 is the gene encoding the ocular lens-specific intrinsic membrane protein MP19. We previously reported finding a single nonconservative G->T transversion in exon two of the Lim2 gene. This mutation was linked to the cataract in the To3 (Total opacity number 3) mouse mutant, confirming Lim2 as an ideal candidate gene for the To3 cataract. The aim of the present study was to substantiate a causative relationship between the mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. To this end a Lim2To3 transgene cassette was engineered and introduced into fertilized normal mouse embryos to test its ability to induce cataractogenic lens development. METHODS: A Lim2 genomic clone was isolated and purified from a murine 129/SvJ genomic library. A restriction endonuclease map of the gene was generated using classical Southern techniques. The murine Lim2 promoter was characterized by transfecting primary chicken lens epithelial cells with Lim2 promoter-CAT reporter constructs and assaying promoter activity and specificity. This genomic clone was then used in conjunction with PCR to generate a Lim2To3 transgene cassette. After sequencing of the PCR engineered portion, the Lim2To3 transgene was then used to generate Lim2To3 transgenic mice via pronuclear injection. Founder mice and their offspring from outcrosses and intercrosses were characterized by ophthalmic examination, PCR and Southern DNA analysis, RT-PCR mRNA analysis, and histology of lens sections. RESULTS: Two mice, from independent microinjections, were identified as positive for presence of the Lim2To3 transgene cassette as well as presence of bilateral congenital cataracts and reduced eye size and mass. One of these founders was incapable of germline transmission of the transgene to offspring and was not characterized further. The other was capable of germline transmission and was characterized as described above. PCR DNA analysis revealed a perfect concordance between presence of the Lim2To3 transgene cassette and congenital cataract in offspring of this founder. Transgenic hemizygotes exhibited cataract and a reduction in eye and lens size and mass, while transgenic "homozygotes" presented with a more severe cataract and microphthalmic reduction in eye and lens size and mass. Southern analysis revealed approximately 2 copies of the transgene cassette integrated into a single chromosomal site in the founder and all hemizygous offspring. RT-PCR analysis revealed a very low ratio of Lim2To3 transgenic mRNA compared to endogenous normal Lim2. Finally, histology revealed that lens development was abnormal in mutant transgenic animals by embryonic day E15. By E19, just prior to birth, gross disorganization of secondary fibers was observed in mutants. CONCLUSIONS: These transgenic experiments firmly establish a causative relationship between the previously identified mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. The low levels of mutant mRNA produced by the transgene cassette as compared to endogenous levels of normal Lim2 mRNA provides evidence that this dominant mutation results in a mutant MP19 protein with altered function rather than simply loss of function.

A hybrid FLEET model for emergency medical service system design.

Covering models have been used to locate emergency services such as ambulance and fire protection systems. As an example, in the late seventies, an analysis of the Baltimore, Maryland fire protection system was conducted with the development and use of a covering model called the Facility Location and Equipment Emplacement Technique (FLEET). The FLEET model combined the location of fire stations with the allocation of primary and special service equipment to the stations. Further, in a recent study of Austin, Texas the ambulance system was restructured based on the use of a covering model. Covering models have also been extended to handle some of the special circumstances involved in emergency service systems. One example is the maximal expected coverage problem (MEXCLP). This paper presents a new covering model which utilizes both the special coverage structure of the MEXCLP and the simultaneous station location and equipment allocation of the FLEET model. Optimal solutions are found using linear and integer programming. Results of the model applied to several planning data sets (including a form of the Austin, Texas planning problem) demonstrate that more concentrated ambulance allocation patterns exist which may lead to easier dispatching, reduced facility costs, and better crew load balancing with little or no loss of service coverage. Tradeoff curves are presented which show that significant reductions in the number of dispatching sites (keeping the number of ambulances constant) can be made without any major changes in service level.

Assignment of the lens intrinsic membrane protein MP19 structural gene to human chromosome 19.

We have isolated and characterized a bovine cDNA clone encoding the bovine lens intrinsic membrane protein, MP19. This cDNA was used as a probe to analyze a panel of Southern blots of human-Chinese hamster somatic cell hybrid DNAs to assign the gene coding for MP19 to its human chromosome. Control human and Chinese hamster DNAs displayed a distinct EcoR1 restriction fragment pattern when hybridized with the bovine MP19 cDNA. When somatic cell hybrid DNAs were restricted with Eco R1 and Southern blots hybridized with the bovine MP19 cDNA, the characteristic human restriction pattern was observed only when human chromosome 19 was present in the hybrid panel. This assignment was confirmed using a human chromosome 19-specific genomic library. A clone from this human chromosome 19-specific library was identified and further characterized. This clone contained a 7.9 kilobase fragment that contained identical DNA sequences with that of the authentic bovine MP19 cDNA, and with a separate human genomic clone containing the MP19 gene.

The human lens fiber-cell intrinsic membrane protein MP19 gene: isolation and sequence analysis.

DNA sequence analysis of overlapping shotgun and restriction fragments have revealed the entire sequence of the human lens fiber cell intrinsic membrane protein MP19 gene (also termed MP17, MP18, and MP20). The 8,056 bp MP19 gene contains 5 exons encoding a mature protein of 173 amino acids, which displayed a very high degree of identity (91%) with that of bovine MP19, deduced from a bovine cDNA sequence. The exon range in size from 52 bases (exon 1) to about 340 bases (exon 5). The introns consist of two large segments (introns B and C) of about 4,700 bases and 1,800 bases, respectively, and two small segments (intron A and D) of about 450 and 250 bases each. Seven Alu family DNA repeats are found within the human MP19 gene. The sequenced gene includes 100 bases of 5' flanking sequence.

Amino acid sequence analysis of proteins in the human corneal stromal cell membrane.

Plasma membrane proteins from human corneal stromal fibroblasts were isolated and separated by two-dimensional polyacrylamide gel electrophoresis. Separated polypeptides were electroeluted onto polyvinylidene difluoride (PVDF) membranes and individual polypeptides were subjected to NH2-terminal amino acid sequence analysis. Of a total of 33 polypeptides sequenced, 26 were found to be blocked at their NH2-terminus. Seven major membrane polypeptides were sequenced and further analyzed. One polypeptide, designated #18, was determined to be homologous to the beta subunit of prolyl hydroxylase/protein disulfide isomerase/thyroid hormone-binding protein. The other six polypeptides were found to have no significant sequence homology with any known polypeptides, as revealed by a protein data base homology search. Polypeptide Bands #90, #102, and #103 were found to have the same NH2-terminal amino acid sequence and the same overall molecular weight, yet separated from one another according to pI. These three polypeptides probably arose from differential posttranslational modification of the same original protein. Synthetic peptides were prepared from the #18 and #19 sequence and antibodies were produced. Immunostaining of cultured human corneal stromal cells and frozen sections of corneas demonstrated that these membrane polypeptides were present in corneal keratocytes, both in vitro and in vivo. Antibody against #18 stained fixed cultured corneal fibroblasts in a very fibrous pattern, with more intense staining in the perinuclear region of the cell, while antibody against #19 stained the cell surface in a much more uniform pattern. In sections of human cornea, both antibodies stained only the keratocytes in the stroma, but they also appeared to stain epithelial cells.

In vitro production of basement membrane collagen by a clonal line of mouse lens epithelial cells.

The basement membrane is a structure of prime importance for the proper functioning of certain organs such as the lens and the kidney. Its inaccessibility and resistance to extraction, however, make an absolute determination of its composition difficult. The establishment of a cell line that synthesizes authentic basement membrane components in vitro would make basement membrane components more easily obtained, and would provide a controlled situation which could be more easily manipulated. In this report, a Balb/C mouse lens epithelial cell line was investigated. The cells were observed to undergo morphological transformations in vitro depending upon the cell density. As cell-cell contacts became prevalent, stellate cells organized into an epithelioid sheet. Later, elongate cells and lentoid bodies predominated in the culture. Thus, morphologically the cells mimicked the in vivo transition of the lens epithelial cells into lens fiber cells. Furthermore, the collagen(s) synthesized by these cells reacted specifically with affinity purified antibody directed against mouse type IV collagen. These morphological and immunological data lend credence to the concept that this lens epithelial cell line is an authentic replica of the in vivo situation.

The human lens intrinsic membrane protein MP70 (Cx50) gene: clonal analysis and chromosome mapping.

We have isolated and characterized a human genomic clone containing the complete coding region of lens intrinsic membrane protein MP70 (Cx50). The coding region of this DNA is completely contained within one exon, as is common of all connexins investigated to date. The size of the Cx50 coding region, from the initiating ATG to the terminating TGA is 1,299 nucleotides, coding for a polypeptide of 432 amino acids and having a translated molecular weight of 48,171 daltons. This Cx50 coding region DNA was used as a probe to analyze a panel of Southern blots of human-Chinese hamster somatic cell hybrid DNAs to assign the gene coding for Cx50 to its human chromosome. Control human and Chinese hamster DNAs displayed a distinct Eco R1 restriction fragment pattern when hybridized with the human Cx50 DNA probe. When somatic cell hybrid DNAs were restricted with Eco R1 and Southern blots hybridized with the human Cx50 DNA probe, the characteristic human restriction pattern was observed only when human chromosome 1 was present in the hybrid panel. Of the other six connexin genes which have previously been assigned to a human chromosome, two of these, Cx37 and Cx40, are also found on chromosome 1.

Molecular cloning and complete nucleotide sequence of the cDNA encoding a bovine lens intrinsic membrane protein (MP19).

Recently, we reported the partial characterization of bovine lens intrinsic membrane proteins having apparent SDS-PAGE derived molecular mass of 19, 21, and 23 kDa, and determined that they contained identical NH2- terminal amino acid sequences for the first 20 amino acids. From this amino acid sequence information, a mixed synthetic oligonucleotide was constructed and used to screen a calf lens lambda gt11 cDNA library in order to isolate and characterize the cDNA coding for this membrane polypeptide(s). Two separate cDNA clones were isolated and sequenced, and were found to have an identical sequence of 883 bases with an open reading frame coding for a polypeptide of 173 amino acids, having a molecular mass of 19,683 Daltons. The first 20 amino acids of the translated sequence were identical to that determined by our laboratory previously, and the last seven amino acids were identical to that recently determined by another laboratory from analysis of the extracted polypeptides, indicating that this cDNA is the authentic molecule coding for MP19.

Expression of c-myc protooncogene in primary cultures of human corneal stromal cells.

We have determined steady-state levels of c-myc mRNA in quiescent and serum-stimulated human corneal stromal cells. Steady-state levels of c-myc mRNA increased 6-fold following 2 hours of serum stimulation over levels observed at quiescence. A parallel increase in the rate of c-myc gene transcription was observed in serum-stimulated cells as compared to quiescent cells, indicating that the abundance of c-myc transcripts in corneal stromal cells during the transition from quiescence to proliferation is regulated mainly at the transcriptional level. These findings indicated that the expression of c-myc gene in human corneal stromal cells is regulated in a cell growth dependent manner in response to serum induction.

Association of alpha-crystallin with actin in cultured lens cells.

The nature of the beaded filaments in the lens fiber cell has been debated for some time. One explanation is that beaded filaments represent an association of alpha-crystallin with actin filaments. By using a double labelling technique that allowed us to view actin filaments and alpha-crystallin in the same cell we have demonstrated that some of the alpha-crystallin in lens cells is indeed associated with actin.