Robert L. Church

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.

cDNA Clones encoding bovine γ-crystallins

Abstract We have determined the nucleotide sequence of two bovine lens γ-crystallin cDNA clones, pBLγII-1 and pBLγIII-1. The 644 bp cDNA insert of pBLγII-1 contains coding information for the entire amino acid sequence of bovine γII-crystallin. The 497 bp cDNA insert of pBLγIII-1 encodes a homologous but different γ-crystallin polypeptide, and appears to lack the coding information for the C-terminal 17 amino acid residues. While the nucleotide and predicted amino acid sequences of the coding regions of the clones show a high degree of homology, the untranslated leader sequences are relatively dissimilar. The leader sequence of pBLγIII-1 is strikingly homologous to a portion of a rabbit immunoglobulin α-heavy chain mRNA.

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.

Regulation of CAD gene expression in mouse fibroblasts during the transition from the resting to the growing state

We have analyzed the steady-state levels of CAD mRNA and ATCase activity in BALB/c 3T3 mouse fibroblasts at quiescence and at various time points following the initiation of serum stimulation. Steady-state levels of CAD mRNA in 3T3 cells following 12 h of serum stimulation increased 10-fold over levels measured at quiescence. In contrast to the observed increase in steady-state levels of CAD mRNA, its rate of transcription increased only 3-fold, suggesting that the expression of CAD gene in these cells is regulated at both the transcriptional and post-transcriptional levels, to a major extent by the latter. These increases in CAD mRNA in serum-stimulated cells were followed by parallel increases in ATCase activity as well. When comparing DNA synthesis [( 3H]thymidine uptake) to the accumulation of CAD mRNA and ATCase activity, it was observed that this accumulation occurred during the mid- to late-G1 phase of the cell cycle. These results suggest that the expression of CAD gene is cell growth dependent and may be a prerequisite to DNA synthesis.

Bovine Lens Membrane Proteins: MP70, MP64, and MP38 are Products of the Same Gene

We have carried out limited microsequence analysis of bovine lens intrinsic membrane proteins having molecular weights of 70, 64, and 38 kD. These three polypeptides all have an identical amino acid terminal sequence, at least for the first 17 amino acid residues, indicating a common origin. When calf lens RNA was hybridized with a labeled antisense oligonucleotide common to the amino acid sequence of these three polypeptides, a single message with an apparent molecular size of 2.6 kb was detected. Together, these results indicate that bovine lens MP70, MP64, and MP38 are products of the same gene and that the lower molecular weight polypeptides are the result of degradation (processing) of lens MP70 at its COOH-terminal end.

Assignment of the mouse alpha A-crystallin structural gene to chromosome 17.

alpha A2-crystallin is one of the major water-soluble proteins of the mammalian lens. Using a cloned cDNA probe coding for mouse alpha A2-crystallin and Southern blot hybridization, DNA isolated from a panel of somatic cell hybrids prepared from mouse fibroblasts or mouse spleen cells fused with Chinese hamster fibroblasts was probed to determine the chromosomal localization of the alpha A2-crystallin structural gene. We have located this gene on mouse chromosome 17.

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.

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.

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.

Regulation of expression of c-myc protooncogene in a clonal line of mouse lens epithelial cells by serum growth factors

Steady-state levels of c-myc mRNA were determined in a clonal line of mouse lens epithelial cells in quiescent and growth-stimulated states. Steady-state mRNA levels for c-myc increased rapidly from an undetectable amount in quiescent cells to the maximum level (8-fold) in growth-stimulated cells. In contrast to its steady-state mRNA levels, its rate of transcription increased by only 3.4-fold in serum-stimulated cells versus quiescent cells, indicating that the abundance of c-myc transcripts in lens epithelial cells during the serum-induced transition from quiescence to proliferation is regulated by both transcriptional and post-transcriptional mechanisms. Serum stimulation in combination with cycloheximide caused superinduction in the steady-state levels of c-myc mRNA in lens epithelial cells. These additive increases in c-myc mRNA levels in the presence of cycloheximide could be due to a decrease in the apparent turnover rate of c-myc mRNA, which, in fact, was observed in actively growing cells. DNA synthesis, as revealed by [3H]thymidine uptake, began 18 h after the addition of serum to quiescent cells and peaked at 24 h. From these results it is concluded that the expression of c-myc gene in mouse lens epithelial cells in response to serum induction is growth dependent.