3 : 5, 1997 <http://www.emory.edu/molvis/v3/steele>|
Received 24 Mar 1997 | Accepted 2 May 1997 | Published 7 May 1997
Identification of a mutation in the MP19 gene, Lim2, in the cataractous mouse mutant To3
Ernest C. Steele, Jr.,1 Stefan Kerscher,2
Mary F. Lyon,2 Peter H. Glenister,2 Jack Favor,3
JianHua Wang,1 and Robert
1Emory Eye Center, Emory University School
of Medicine, Department of Ophthalmology, Atlanta, GA, USA
2MRC Mammalian Genetics Unit, Harwell, Didcot, Oxfordshire OX11 ORD, United Kingdom
3Institute of Mammalian Genetics, GSF - National Research Center for the Environment and Health, D-85758 Neuherberg, Germany
Robert L. Church
Emory Eye Center
1365B Clifton Rd, NE
Atlanta, GA 30322 (USA)
Phone: (404) 778-4101
FAX: (404) 778-2232
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.
MP19 (lens intrinsic membrane protein, 19 kDa) is the second most abundant integral membrane protein within vertebrate ocular lens fiber cells (1,2). MP19 has been shown to be localized to tight junctional domains of differentiated lens fiber membranes (2-6). The protein has four predicted transmembrane domains and has been demonstrated to be phosphorylated (2, 7-9) and to bind calmodulin in vitro (2, 7, 10, 11). These features are characteristic of functional proteins that are regulated or modulated. To date, no clearly defined structural or functional role within the lens has been elucidated for MP19. However, there is now a preponderance of evidence that a genetic lesion in Lim2 (lens intrinsic membrane protein #2), the gene encoding MP19, is the underlying molecular defect in the cataractous mouse mutant, To3 (total opacity of lens #3)(18). This new evidence suggests an important role for MP19 in both normal lens development and cataractogenesis.
To3, originally named ENU-288, was produced and characterized in a set of experiments testing the efficacy of ENU (ethylnitrosourea) as a mouse spermatogonial mutagen (12). This mutant was further characterized in extensive outcrossing experiments, and the phenotype was demonstrated to segregate in a simple Mendelian fashion with full penetrance, having no effect on viability or fertility (13). The To3 mutant phenotype is both hereditary, being expressed in a semi-dominant fashion, and congenital. Mice heterozygous or homozygous for the To3 mutation have total opacity of the lens with a dense cataract. In addition, the To3/To3 homozygotes exhibit microphthalmia, abnormally small eyes. Histological sections of the eyes of homozygotes show that the lens is vacuolated and the fibers are grossly disorganized (Figure 1). In the central region, fibers lose their nuclei, but are not regularly arranged. Disorganized secondary fibers retaining nuclei are also present (Figure 1b). In homozygotes, but not heterozygotes, the lens capsule ruptures posteriorly with lens material leaking into the vitreous chamber (Figure 1c), and in some cases the lenses were vacuolated. The vitreous chamber was abnormally small, and the eyes appeared shrunken. The development of the abnormality has not yet been studied. Rupture of the lens is also found in various other mouse cataracts, including lens rupture, lr, (14), and rupture of lens cataract, rlc, (15). Similarly, microphthalmia accompanies cataract in other mouse mutants, including alleles of dominant cataract, Cat (16) and of dominant cataract 2, Cat2 (17).
Recently, the To3 cataract was mapped to a position approximately 7.3 cM proximal to the coat color marker, p, on mouse chromosome 7 and the name To3 (for Total Opacity #3) was proposed for the genetic locus (18). Using a bovine Lim2 (MP19) cDNA probe, that possesses greater than 90% nucleotide identity with the mouse sequence, the Lim2 gene was recently mapped to the same region of mouse chromosome 7 as the To3 cataract (approximately 8 cM proximal to p) (19). Although To3 and Lim2 have not been directly mapped against one another, the closeness of their apparent locations on mouse chromosome 7 makes Lim2 a likely candidate gene in the To3 mutant. This is further supported by the fact that the only other lens specific genes known to map to this region of mouse chromosome 7, Zfp61 and Zfp63 (both of which are transcription factors), map too proximally to be candidates for To3 (20).
Based upon the above evidence, we have screened the Lim2 genes of both the To3 mutant and normal parental strains in an attempt to detect an alteration in the Lim2 gene of the To3 mutant that was associated and consistent with the observed phenotype. In this communication we report the identification of a single base change within the first coding exon of the To3 mutant Lim2 gene that results in a single amino acid substitution within the MP19 protein. This new evidence indicates that MP19 may play an important role in both lens development and cataractogenesis and warrants further investigation into the precise nature of its role within the ocular lens.
Materials and Methods
Isolation of murine genomic DNA's
High molecular weight genomic DNA was isolated from 200 mg of frozen mouse spleens from normal parental mouse strains and heterozygous and homozygous To3 mice according to standard methods using Proteinase K digestion of cells followed by organic extraction and precipitation of DNA (21). The DNA's were quantitated using a UV spectrophotometer and were analyzed on agarose gels to confirm high molecular weight and lack of degradation.
PCR amplification of murine Lim2 gene fragments
An approximately 3.8 kb fragment spanning from about 130 bases upstream of the transcription start site through the end of exon 3 (Fragment 1 in Figure 2) was amplified using rT.th. DNA polymerase (Perkin-Elmer) and the H19P-132/MP19-386 primer pair (See Table 1 for sequences) in a PTC-200 DNA Engine (MJ Research, Watertown, MA). Briefly, PCR was carried out using 100 ng genomic DNA as template, the supplied buffer, 200 µM each dNTP, and 25 pmoles of each primer using a "hot start" technique. The following cycling conditions were used: 120s initial denaturation at 94oC followed by 40 cycles of 60s at 93oC, 60s at 58oC, and 360s at 72oC followed by 10 min at 72oC.
Another fragment, spanning from the beginning of exon 3 through the end of exon 4 (Fragment 2 in Figure 2, approximately 1.9 kb) was amplified as above using the MP19-239/MP19-524 primer pair (See Table 1 for the sequences). PCR was carried out as for Fragment 1 above using the following cycling conditions: 120s initial denaturation at 94oC followed by 40 cycles of 60s at 93oC, 60s at 60oC, 120s at 72oC followed by 10 min at 72oC.
A third fragment, spanning from the beginning of exon 4 through to about 100 bp past the translational stop site (Fragment 3 in Figure 2, approximately 500 bp) was amplified using P.fu. DNA polymerase (Stratagene, La Jolla, CA) and the 129X5S2/129X5A2 primer pair (See Table 1 for sequences). PCR was carried out using 100 ng genomic DNA as template, the supplied buffer, 200 µM each dNTP, and 100 pmoles of each primer with a "hot start" technique. The following cycling conditions were used: 120s at 94oC initial denaturation followed by 40 cycles of 60s at 94oC, 60s at 58oC, 60s at 72oC followed by 10 min at 72oC.
PCR of Lim2 exon 2 fragments for confirmation
An approximately 2.2 kb fragment spanning exon 2 (Fragment 4 in Figure 2) was amplified using rT.th. DNA polymerase and the M19TG-S2/M19TG-A3 primer pair (See Table 1 for sequences). PCR was carried out as for Fragments 1 and 2 above using the following cycling conditions: 120s at 940C initial denaturation followed by 39 cycles of 60s at 93oC, 60s at 60oC, and 240s at 72oC followed by 10 min at 72oC. Fragment 4 PCR products were digested with BstEII restriction endonuclease (Life Technologies, Gaithersburg, MD) for 30 min at 37oC, then electrophoresed on a 2% agarose gel stained with ethidium bromide, and visualized using the Gel Doc 1000 apparatus and Molecular Analyst Software (Bio-Rad Laboratories, Hercules, CA).
Fragments 1 and 2 described above were subcloned into the pCRII vector (Invitrogen, Carlsbad, CA). These constructs were then used as template in cycle sequencing reactions using Cy5-labeled oligonucleotide primers and the Cy5 AutoCycle Sequencing Kit (Pharmacia Biotech, Piscataway, NJ). These were then electrophoresed and analyzed using an ALFexpress automated DNA Sequencer (Pharmacia Biotech, Piscataway, NJ). Fragments 3 and 4 were purified on a preparative agarose gel and then sequenced directly without subcloning using the same reagents and methods.
Nucleotide sequence and predicted amino acid sequence analysis was performed using DNASIS and PROSIS software (Hitachi America, Ltd.). Secondary structure analyses were performed using Macimdad (Molecular Applications Group) and PROSIS software.
Results and Discussion
Before undertaking the screening of the Lim2 gene in To3, the type of mutagenesis used to create this mutant, the alkylating agent N-ethyl-N-nitrosourea (ENU), was considered. ENU is a highly effective mutagen for mouse spermatogonial stem cells (22) that was predicted from studies in Drosophila (23) to induce single-base-pair changes in mammalian DNA, resulting in the recovery of intragenic mutations. The available data on the genetic and molecular nature of ENU-induced specific locus mutations in the mouse are consistent with this notion (24-26).
Therefore, it was sound to hypothesize that the mutation in the To3 mutant would be a single-base-pair mutation. Furthermore, the dominant nature of the mutant phenotype implied that the mutation would probably result in a protein with an altered structure and/or function. There were three possible locations for a mutation that would be expected to alter a protein's structure and/or function:
As there was no method of predicting the odds of any of these types of mutations being more likely than the other, all three were screened for.
In order to screen the entire coding region, including the splice sites and the translation termination signal, the PCR primers shown in Table 1 were designed to amplify three large overlapping fragments encompassing different portions of the coding region as indicated in Figure 2. PCR was carried out on genomic DNA isolated from both normal parental strains as well as heterozygous and homozygous mutants. Both strands of the coding regions were then sequenced using oligonucleotides corresponding to sequences internal to these amplified fragments.
The only reproducible sequence change discovered was a single G -> T transversion within the first coding exon (exon 2) of the Lim2 gene. The sequence comparison for this region is shown in Figure 3. The sequence shown in Figure 3a is the antisense strand and, therefore, shows a C -> A transversion. This DNA sequence alteration results in the creation of a Bst EII restriction endonuclease cleavage site within the DNA as well as the substitution of the normally encoded glycine by a valine at amino acid 15 within the polypeptide sequence as shown in Figure 3b. The Bst EII site was used as shown in Figure 4 to confirm the presence of the DNA sequence alteration in the To3/To3 and To3/+ DNA, and its absence in the +/+ DNA.
Using predictive secondary structure algorithms (27,28), the glycine -> valine substitution was observed to result in a turn within the alpha helix of the first transmembrane spanning domain of the MP19 polypeptide. A graphical representation of this predicted change in the protein conformation was produced using Macimdad Software and is shown in Figure 5. Though speculative, such a disruption of the protein's normal folding pattern could interfere with normal insertion of the protein into fiber cell membranes and thus account for the defects observed in the mutant lenses. However, no physical structural analyses of normal and mutant MP19 protein have been conducted to confirm this theoretical model.
As Figures 3 and 4 illustrate, the identified mutation is linked to the observed cataractous phenotype of the To3 mutant. The mutation is also consistent with the observed phenotype for the following reasons:
It is of note that our results with the To3 mutant bear a great deal of similarity to that of another murine mutant, Lop (for lens opacity). Lop is another autosomal semi-dominant, congenital, bilateral cataract. The Lop cataract has recently been shown to be the result of a single base pair transversion in the coding region of Mip, the gene encoding the most abundant lens specific integral membrane protein MP26. This nucleotide change results in the nonconservative substitution of proline for alanine at amino acid 51 (30). The evidence supports the hypothesis that the mutation alters the proper folding of the protein, resulting in its improper trafficking. Instead of being inserted into the membranes of the fiber cells, the mutant MP26 protein appears to accumulate in the endoplasmic reticulum. It is tempting to hypothesize that the same phenomenon might be occurring with the MP19 protein in the To3 mutant. It is equally tempting to hypothesize that MP19 may play a role similar to, or perhaps even interactive with, MP26, as both proteins colocalize to the junction regions of the lens membranes and appear to result in similar phenotypes when one or the other is mutated.
Previously, immunofluorescent studies of MP19 in lens fiber cells colocalized MP19 with the gap junction protein Cx46 in a restricted area 0.5 mm to 1.0 mm into the lens(4). It is, therefore, another possibility that MP19 may serve a role similar to or interactive with this protein. These data provide evidence of a possible role for MP19 in mammalian lens fiber junctional formation or organization.
While the above correlations add strong support to the hypothesis that a genetic lesion in the Lim2 gene of the To3 mutant is the causative factor for the cataractous phenotype, they are by no means proof. It remains a possibility, albeit highly unlikely, that there exists an unidentified lens specific gene adjacent to Lim2 which also bears a genetic lesion and is actually the cause of the phenotype in the To3 mutant. In order to prove a causative relationship between the identified mutation and the cataractous phenotype, a transgene would have to be generated, by engineering this same mutation into a normal murine Lim2 gene, and used in classical transgenic experiments.
In conclusion, these data support the hypothesis that a genetic lesion in the Lim2 gene is the causative factor for the cataractous phenotype of the To3 mouse mutant. The identified G -> T transversion in exon 2 results in a nonconservative substitution of valine for glycine at amino acid position 15 in the first transmembrane domain of the encoded MP19 polypeptide. This amino acid substitution may alter the normal conformation, and thereby any associated function, of the encoded MP19 polypeptide. As the role of MP19 in the ocular lens remains undetermined and no immunohistochemical analyses of the To3 lenses have been conducted, it is impossible to predict whether the mutant MP19 is a gain of function or a loss of function allele. One hypothesis is that To3 represents a gain-of-function mutation involving protein conformation defect or even cytotoxic effects in the fiber cells. An alternative hypothesis is that To3 represents a loss-of-function allele such that a reduced concentration below a threshold concentration results in cataract formation. Transgenic experiments and immunohistochemical analyses should provide final proof or disproof of these hypotheses. If the mutation in the Lim2 gene proves to be the underlying molecular defect in the To3 mutant, the Lim2 gene becomes another candidate for certain types of hereditary cataract in the human population.
This work was supported in part by National Institutes of Health grants R01 EY11516, T32 EY07092, P30 EY06360, C06 EY06307, RO1 EY10321, the Knights Templar Educational Foundation of Georgia, Inc., and a Departmental Grant from Research to Prevent Blindness, Inc.
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