Current hYe
Research
Volume I I numhcr 5 1992. 421 -424
Assignment of the lens intrinsic membrane protein MP19 structural gene to human chromosome 19 Robert L.Church and JianHua Wang
Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 02/05/15 For personal use only.
Emory University School of Medicine, Department of Ophthalmology, 1327 Clifton Road N.E., Atlanta, GA 30322, USA
ABSTRACT 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 MP 19 to its human chromosome. Control human and Chinese hamster DNAs displayed a distinct EcoRl restriction fragment pattern when hybridized with the bovine MP 19 cDFA. When somatic cell hybrid DNAs were restricted with Eco R 1 and Southern blots hybridized with the bovine MPI 9 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 MP 19 gene.
In this communication, we present the localization of MP19 to its human chromosome, using a commercially available human-Chinese hamster somatic cell hybrid panel (BIOS Corporation), and a human chromosome-specific genomic DNA library. Portions of this work have appeared in abstract form
(13).
MATERIALS AND METHODS
cd!llnesaadsomaticdhvbnds The somatic cell hybrid panel used in these studies was generated from Chinese hamster ovary (CHO-KI) cells and normal human B-lymphocytes, as described by Carlock, et al. (14). This panel was prepared by BIOS Corporation (New Haven, CT) and consists of 18 separate somatic cell hybrids containing various combinations of human chromosomes, and standard parental lines of Chinese hamster and human origin. This panel has previously been used to map 16 different human gene sequences by others ( 15-17). Southern blots were prepared from total genomic DNA isolated from each of the hybrid and parental lines. Eight pg of DNA from these hybrids and parents were digested with EcoRl and fractionated in 0.8% agarose gels for 320 volt-hours. The fractionated DNAs were then denatured and transferred to a neutral nylon membrane and crosslinked to the membrane by exposure to UV irradiation (1 8). The DNA panel was probed with 32P-labeled lens MP 19 cDNA (883 base pairs, ref. 8). Purified probe was labeled and Southern blots screened as described previously (1 9).
INTRODUCTION The mammalian lens fiber cell membrane contains five major proteins, ranging from 70 kDa to 19 kDa in size (1,2). The specific function of these proteins is, at present, unknown. However, all of these protein molecules may be involved in lens cell communication or transport (3,4). Some of these membrane proteins have been shown to be involved with cataractogenesis (5-7). The second most abundant intrinsic membrane protein of the lens fiber cell is MP19 (Major lens Erotein) having a molecular weight of 19.5 kDa, determined from it's deduced amino acid sequence (8). This protein appears to contain four transmembrane domains (8), is a substrate for CAMP-dependent protein kinase (9, 10) and proteins kinase C (1 I), and binds with calmodulin (9, 12). Taken together, these indicate that MP 19 probably functions in some way as a junctional component, possibly involved with lens cell communication. Due to its potentially important communication role in lens membrane function, and because it has bcen shown to be involved with cataractogcncsis, furthcr knowledge of its genetic make-up and chromosomal localization is of interest. ~~~
~.
Humangsmisllbranes Two separate human genomic libraries were used in these studies. The first library was generated from the human lung fibroblast cell line W138 (M46204, Stratagene, La Jolla, CA) and was used to isolate a human genomic clone of MP19, using the bovine MP 19 cDNA as a probe ( I 3). The second library ~
Received on November 6. 1991: accepted on March 23, 1992 ~~
~
~
0 Oxford University Press
42 1
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 02/05/15 For personal use only.
was generated from a human-Chinese hamster somatic cell hybrid line containing only chromosome 19 as human genetic material (American Type Culture Collection, ATCC#57766). Human chromosome 19 was purified from this hybrid line by flow-sorting and the DNA was partially digested with Mbo 1, and DNA fragments cloned into the Bam HI site of Charon 40. Estimated purity of human chromosome 19 in this library is 84%, with the impurity consisting of Chinese hamster DNA. This library was used to confirm the MP19 chromosome assignment. DNA library screening and Southern analysis of cloned DNA fragments were carried out as described in a previous communication (19). RESULTS AND DISCUSSION Human and Chinese hamster genomic DNAs digested with EcoRl and separated on agarose demonstrates distinct and specific patterns when hybridized with the bovine MP 19 cDNA (Figure 1, lanes 1 & 11 in the upper panel, and lanes 12 & 22 in the lower panel). Hybrid clones containing a positive human MP 19 signal are shown in Figure 1, upper panel #s 7 & 8, and lower panel #s 15. 17, 18, & 19. All other hybrid clones in Figure 1 are negative for the presence of the human MP 19 gene. The human MP 19 structural gene segregates with complete concordancy with human chromosome 19 (Table I), and can be tentatively be assigned to this human chromosome. Further confirmation of this assignment was obtained by using a DNA library constructed specifically from human chromosotne 19. Using this library, we were able to identify a positive clone by screening the library with the bovine MP 19 cDNA. This clone contained a human DNA insert of about 7.9 kb that, upon digestion with EcoR I , yielded two fragments of approximately 1.8 and 6 kb, very similar in size to those obtained from EcoRl digestion of an MP 19-positive clone derived from another human genomic library containing the entire human genome (1 3). Partial sequence analysis of the 1.8 kb fragment from the human chromosome 19-specific library demonstrated a very high degree of identity with the coding region of the bovine MPI 9 cDNA and an identical sequence with a 2.5 kb fragment isolated from the total human genomic library (Table 2). Neither the nucleotide sequence nor the deduced amino acid sequence presented above (Table 2) demonstrated identity above 35% when these sequences were screened against all presently known protein or DNA sequences in the databases (GenBank or Protein Identification Resource). Taken together, these data
422
Figure 1. Southern blot analysis of EcoRl digested human, Chinese hamster. and human-Chinese hamster somatic cell hybrid DNAs using the 883 base-pair bovine lens MP 19 cDNA probe. Lane 1 in the upper and lane 12 in the lower blot is standard human DNA and lane 1 1 in the upper and lane 22 in the lower blot is standard Chinese Hamster DNA. Marker "0" in the figure indicates the origin of the upper and lower panels. Band A designates the human-specific DNA fragment of about 6 kb. Hybrids in lanes 7 and 8 in the upper blot and hybrids 15, 17, 18, and 19 in the lower blot are positive for the human MP19 gene. Lane numbers and corresponding hybrid number from Table 1 are as follows: lane 2=hybrid 937,3=854,4=507, 5=983,6=1079,7=1006,8=756,9=904,10=909, 13=967, 14=862.15=860,16=1049,17=683,18=867,19=750,20=212, and 21=734.
Current Eye Research Table 1 . Segregation of MP19 gene and human chromosomes in human-Chinese hamster somatic cell hybrids. Human Chromosome
Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 02/05/15 For personal use only.
Hybrid
MP19
937 854 507 983 1079 I006 756 904 909 967 862 860 1049 683 867 750 212 734
-
+ +
Concordanthybrids Discordant hybrids Discordancy (%)
1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 X Y
+ - + - -
+
- - - -
45%
- -
- - + - -
12 1 1 1 1 13 6 12 14 1 1 1 1 12 1 1 12 16 13 12 9 9 12 18 12 14 12 11 10 6 7 7 5 1 2 6 4 7 7 6 7 6 2 5 6 9 9 6 0 6 4 6 7 8 33 39 39 28 67 33 22 39 39 33 39 33 3 28 33 50 50 33 0 33 22 33 29 44
Percentage numbers in the panel are the percent of the cell population containing the noted chromosome.
Table 2. Partial DNA and deduced amino acid sequence of the coding region of bovine MP 19 compared to the equivalent sequences obtained from a human total genomic DNA library and chromosome 19-specific library isolated using the bovine h4P19 cDNA probe.
66 bovineMP19cDNA
ATG TAC AGC l T C
ATG GGA GGC GGC CTC
TTC
676 human genomic
ATG TAC AGC 'ITC
ATG GGT GGX GGC CTG TTC
in
ATG GGT GGT GGC CTG TTC MET GLY GLY GLY LEU PHE
chromosome 19 aminoacidsequence
AT'; TAC AGC 'ITC MET TYR SER PHE
bovineMPlYcDNA
TGT GCC TGG GTG GGG ACC ATC
96 CTC
CTG GTG
CTC
CTG GTG
TGT GCC TGG GTG GGG ACC ATC CTC CYS ALA TRP VAL GLY THR ILE LEU
CTG GTG LEU VAL
706 human genomic
TGT GCC TGG GTG GGG ACC ATC
40 chromosome 19 aminoacidsequence
Numbers above DNA sequences indicate position of basc with rcspect to the 5'-end of the clone. Underlined bases indicate differences between the human sequences and the bovine cDNA. confirm the localization of the lcns MP19 gene to human chromosome 19. This is the second of the lens membrane intrinsic proteins to
be mapped to its specific human chromosome. MP26,the major lens intrinsic membrane protein has previously been mapped to human chromosome 12by Sparkes, d.(20). In the lens, the
423
Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 02/05/15 For personal use only.
Current Eye Research majority of the human crystallins have now been mapped, as well as two membrane proteins. To date, no correlation of the assigned genes for either crystallins or membrane proteins have been made with the various types of congenital cataract in man (2 1,22). Although it appears from the present gene assignment that the MP 19 gene is not associated with any of the presently known congential cataracts (these have been associated with human chromosomes 1,2, 14, 16, and X), it will be of interest to investigate other lens disorders and cataracts to determine if MP19 is associated in any way with the cataractous condition. ACKNOWLEDGEMENTS This work was supported in part by National Institutes of Health grants R01 EY08616, P30 EY06360, and C06 EY06307 and a Departmental Grant from Research to Prevent Blindness, Inc. CORRESPONDING AUTHOR Robert L. Church, Ph.D., Emory University School of Medicine, Department of Ophthalmology, 1327 Clifton Road, N.E.. Atlanta, Georgia 30322 REFERENCES 1 . Rao, G.N., Gutekunst, K.A. and Church, R.L. (1989) Bovine lens 23,2 1 and 19 kDa intrinsic membrane proteins have an identical amino-terminal amino acid 483-486. sequence. FEBS Lett. 2. Rao, G. N., Gutekunst, K.A. and Church, R.L. (1990) Bovine lens membrane uroteins: MP70. MP64. and MP38 are products of the same gene. Ophthal. Res. 2,166-172. 3. Bok, D., Dockstader, J. and Horwitz, J. (1 982) Immunocytochemical localization of the lens main intrinsic polypeptide (MIP 26) in communicating junctions. J. Cell Biol. =,213-220. 4. Gruijters, W.T.M., Kistler, J., Bullivant, S. and Goodenough, D.A. (1 987) Immunolocalization of MP70 in lens fiber 16-17 nm intercellular junctions. J. Cell Biol. M,565-572. 5 . Roy, D., Spector, A. and Famsworth, P.N. (1979) Human lens membrane: Comparison of the major intrinsic polypeptides from young and old lenses isolated by a new methodology. Exp. Eye Res. 28,353-358. 6. Gamer, W.H., Gamer, M.H. and Spector, A. (1981) Gamma-crystallin, a major cytoplasmic polypeptide disulfide linked to mebrane proteins in human cataract. Biochem. Biophys. Res. Commun. eS,439-447. 7. Kadama, T, and Takemoto, L. (1988) characterization of dusulfide-linked crystallins associated with human cataractous lens membranes. Invest. Ophthalmol. Vis. Sci. 29. 145-149. 8. Gutekunst, K.A., Rao, G.N. and Church, R.L. (1990) Molecular cloning and complete nucleotide sequence of the cDNA encoding a bovine lens intrinsic membrane protein (MP19). Curr. Eye Res. 9,955-961. 9. Louis, C.F., Johnson, R., Johnson, K. and Turnquist, J.
m,
424
(1 985) Characterization of the bovine lens plasma
membrane substrates for CAMP-dependent protein kinase. Eur. J. Biochem. Ira,279-286. 10. Garland, D. and Russell, P. (1985) Phosphorylation of lens fiber cell membrane proteins. Proc. Natl. Acad. Sci., USA 82,653-657. 11. Lampe, P.D., Bazzi, M.D., Nelsestuen, G.L. and Johnson, R. (1986) Phosphorylation of lens intrinsic membrane proteins by protein kinase C. Eur. J. Biochem. 114.35 1-357. 12. Mulders, J.W.M., Voorter, C.E., Lamers, C., de HaardHoekman, W.A., Montecucco, C., Van de Ven, W.J.M., Bloemendal, H. and de Jong. W.W.W. (1988) MP17, a fiber-specific intrinsic membrane protein from mammalian eye lens. Current Eye Res. 2,207-2 19. 13. Wang, J-H., Church, R.L. and Neckelmann, N. (1991) Isolation and characterization of human genomic clones coding for lens intrisic membrane protein MP 19 and mapping to its human chromosome. Invest. 971. Ophthalmol. Vis. Sci. ,14. Carlock, L.R., Smith, D. and Wasmuth, J.J. (1986) Genetic counterselective procedure to isolate interspecific cell hybrids containing single human chromosomes: Construction of cell hybrids and recombinant DNA libraries specific for human chromosomes 3 and 4. Somatic Cell Mol. Genet. 163-174. 15. Eubanks, J., Smith, B., Wasmuth, J.J., Kouri, R.E., Brown, J.H. and Evans, G.A. (1989) Mapping of a conserved G protein-coupled receptor-like gene on human chromosome 19. Cytogenet. Cell Genet. 995. 16. Kouri, R.E., Lewis, M., Barker, D.F., Dietz-Band, J.N., Nguyen, K.N., McLemore, T. and Wasmuth, J.J. (1 989) Mapping 14 human gene sequences with a commercially available somatic cell hybrid panel. 1025. Cytogenet. Cell Genet. 17. Pascoe, L., Kouri, R.E. and White, P.C. (1989) A new gene family on chromosome 5 . Cytogenet. Cell Genet. 1058. 18. Southern, E.M. (1 975) Detection of specific sequcnces among DNA fragments separated by gel electrophoresis. J. Mol. Biol. %,503-5 17. 19. Kaye, N.W., Chung, A.E., Lalley, P.A., Durkin, M.E., Phillips, S.L. and Church, R.L. (1 990) Gene mapping of mouse laminin A and B2 subunits using mouse-Chinese hamster somatic cell hybrids. Somatic Cell Mol. Genet. 16, 599-603. 20. Sparkes, R.S., Mohandas, T., Heinzmann, C., Gorin, M.B., Horwitz, J., Law, M.L., Jones, C.A. and Bateman, J.B. (1986) The gene for the major intrinsic protein (MIP) of the ocular lens is assigned to human chromosome 12cenq14. Invest. Ophthalmol. Vis. Sci. 22, 1351-1354. 21. Bateman, J.B., Spence, M.A., Marazita, M.L. and Sparkes, R.S. (1986) Genetic linkage analysis of autosomal dominant congenital cataracts. Am. J. 2 18-225. Ophthalmol. J.Ql, 22. Barrett, D.J., Sparkes, R.S., Gorin, M.B., Bhat, S.P., Spence, M.A., Marazita, M.L. and Bateman. J.B. (1988) Genetic linkage analysis of autosomal dominant congenital cataracts with lens-specific DNA probes and polymorphic phenotypic markers. Ophthalmology 41,538544.
u,
w,
w,
w,
Research
Volume I I numhcr 5 1992. 421 -424
Assignment of the lens intrinsic membrane protein MP19 structural gene to human chromosome 19 Robert L.Church and JianHua Wang
Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 02/05/15 For personal use only.
Emory University School of Medicine, Department of Ophthalmology, 1327 Clifton Road N.E., Atlanta, GA 30322, USA
ABSTRACT 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 MP 19 to its human chromosome. Control human and Chinese hamster DNAs displayed a distinct EcoRl restriction fragment pattern when hybridized with the bovine MP 19 cDFA. When somatic cell hybrid DNAs were restricted with Eco R 1 and Southern blots hybridized with the bovine MPI 9 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 MP 19 gene.
In this communication, we present the localization of MP19 to its human chromosome, using a commercially available human-Chinese hamster somatic cell hybrid panel (BIOS Corporation), and a human chromosome-specific genomic DNA library. Portions of this work have appeared in abstract form
(13).
MATERIALS AND METHODS
cd!llnesaadsomaticdhvbnds The somatic cell hybrid panel used in these studies was generated from Chinese hamster ovary (CHO-KI) cells and normal human B-lymphocytes, as described by Carlock, et al. (14). This panel was prepared by BIOS Corporation (New Haven, CT) and consists of 18 separate somatic cell hybrids containing various combinations of human chromosomes, and standard parental lines of Chinese hamster and human origin. This panel has previously been used to map 16 different human gene sequences by others ( 15-17). Southern blots were prepared from total genomic DNA isolated from each of the hybrid and parental lines. Eight pg of DNA from these hybrids and parents were digested with EcoRl and fractionated in 0.8% agarose gels for 320 volt-hours. The fractionated DNAs were then denatured and transferred to a neutral nylon membrane and crosslinked to the membrane by exposure to UV irradiation (1 8). The DNA panel was probed with 32P-labeled lens MP 19 cDNA (883 base pairs, ref. 8). Purified probe was labeled and Southern blots screened as described previously (1 9).
INTRODUCTION The mammalian lens fiber cell membrane contains five major proteins, ranging from 70 kDa to 19 kDa in size (1,2). The specific function of these proteins is, at present, unknown. However, all of these protein molecules may be involved in lens cell communication or transport (3,4). Some of these membrane proteins have been shown to be involved with cataractogenesis (5-7). The second most abundant intrinsic membrane protein of the lens fiber cell is MP19 (Major lens Erotein) having a molecular weight of 19.5 kDa, determined from it's deduced amino acid sequence (8). This protein appears to contain four transmembrane domains (8), is a substrate for CAMP-dependent protein kinase (9, 10) and proteins kinase C (1 I), and binds with calmodulin (9, 12). Taken together, these indicate that MP 19 probably functions in some way as a junctional component, possibly involved with lens cell communication. Due to its potentially important communication role in lens membrane function, and because it has bcen shown to be involved with cataractogcncsis, furthcr knowledge of its genetic make-up and chromosomal localization is of interest. ~~~
~.
Humangsmisllbranes Two separate human genomic libraries were used in these studies. The first library was generated from the human lung fibroblast cell line W138 (M46204, Stratagene, La Jolla, CA) and was used to isolate a human genomic clone of MP19, using the bovine MP 19 cDNA as a probe ( I 3). The second library ~
Received on November 6. 1991: accepted on March 23, 1992 ~~
~
~
0 Oxford University Press
42 1
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 02/05/15 For personal use only.
was generated from a human-Chinese hamster somatic cell hybrid line containing only chromosome 19 as human genetic material (American Type Culture Collection, ATCC#57766). Human chromosome 19 was purified from this hybrid line by flow-sorting and the DNA was partially digested with Mbo 1, and DNA fragments cloned into the Bam HI site of Charon 40. Estimated purity of human chromosome 19 in this library is 84%, with the impurity consisting of Chinese hamster DNA. This library was used to confirm the MP19 chromosome assignment. DNA library screening and Southern analysis of cloned DNA fragments were carried out as described in a previous communication (19). RESULTS AND DISCUSSION Human and Chinese hamster genomic DNAs digested with EcoRl and separated on agarose demonstrates distinct and specific patterns when hybridized with the bovine MP 19 cDNA (Figure 1, lanes 1 & 11 in the upper panel, and lanes 12 & 22 in the lower panel). Hybrid clones containing a positive human MP 19 signal are shown in Figure 1, upper panel #s 7 & 8, and lower panel #s 15. 17, 18, & 19. All other hybrid clones in Figure 1 are negative for the presence of the human MP 19 gene. The human MP 19 structural gene segregates with complete concordancy with human chromosome 19 (Table I), and can be tentatively be assigned to this human chromosome. Further confirmation of this assignment was obtained by using a DNA library constructed specifically from human chromosotne 19. Using this library, we were able to identify a positive clone by screening the library with the bovine MP 19 cDNA. This clone contained a human DNA insert of about 7.9 kb that, upon digestion with EcoR I , yielded two fragments of approximately 1.8 and 6 kb, very similar in size to those obtained from EcoRl digestion of an MP 19-positive clone derived from another human genomic library containing the entire human genome (1 3). Partial sequence analysis of the 1.8 kb fragment from the human chromosome 19-specific library demonstrated a very high degree of identity with the coding region of the bovine MPI 9 cDNA and an identical sequence with a 2.5 kb fragment isolated from the total human genomic library (Table 2). Neither the nucleotide sequence nor the deduced amino acid sequence presented above (Table 2) demonstrated identity above 35% when these sequences were screened against all presently known protein or DNA sequences in the databases (GenBank or Protein Identification Resource). Taken together, these data
422
Figure 1. Southern blot analysis of EcoRl digested human, Chinese hamster. and human-Chinese hamster somatic cell hybrid DNAs using the 883 base-pair bovine lens MP 19 cDNA probe. Lane 1 in the upper and lane 12 in the lower blot is standard human DNA and lane 1 1 in the upper and lane 22 in the lower blot is standard Chinese Hamster DNA. Marker "0" in the figure indicates the origin of the upper and lower panels. Band A designates the human-specific DNA fragment of about 6 kb. Hybrids in lanes 7 and 8 in the upper blot and hybrids 15, 17, 18, and 19 in the lower blot are positive for the human MP19 gene. Lane numbers and corresponding hybrid number from Table 1 are as follows: lane 2=hybrid 937,3=854,4=507, 5=983,6=1079,7=1006,8=756,9=904,10=909, 13=967, 14=862.15=860,16=1049,17=683,18=867,19=750,20=212, and 21=734.
Current Eye Research Table 1 . Segregation of MP19 gene and human chromosomes in human-Chinese hamster somatic cell hybrids. Human Chromosome
Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 02/05/15 For personal use only.
Hybrid
MP19
937 854 507 983 1079 I006 756 904 909 967 862 860 1049 683 867 750 212 734
-
+ +
Concordanthybrids Discordant hybrids Discordancy (%)
1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 X Y
+ - + - -
+
- - - -
45%
- -
- - + - -
12 1 1 1 1 13 6 12 14 1 1 1 1 12 1 1 12 16 13 12 9 9 12 18 12 14 12 11 10 6 7 7 5 1 2 6 4 7 7 6 7 6 2 5 6 9 9 6 0 6 4 6 7 8 33 39 39 28 67 33 22 39 39 33 39 33 3 28 33 50 50 33 0 33 22 33 29 44
Percentage numbers in the panel are the percent of the cell population containing the noted chromosome.
Table 2. Partial DNA and deduced amino acid sequence of the coding region of bovine MP 19 compared to the equivalent sequences obtained from a human total genomic DNA library and chromosome 19-specific library isolated using the bovine h4P19 cDNA probe.
66 bovineMP19cDNA
ATG TAC AGC l T C
ATG GGA GGC GGC CTC
TTC
676 human genomic
ATG TAC AGC 'ITC
ATG GGT GGX GGC CTG TTC
in
ATG GGT GGT GGC CTG TTC MET GLY GLY GLY LEU PHE
chromosome 19 aminoacidsequence
AT'; TAC AGC 'ITC MET TYR SER PHE
bovineMPlYcDNA
TGT GCC TGG GTG GGG ACC ATC
96 CTC
CTG GTG
CTC
CTG GTG
TGT GCC TGG GTG GGG ACC ATC CTC CYS ALA TRP VAL GLY THR ILE LEU
CTG GTG LEU VAL
706 human genomic
TGT GCC TGG GTG GGG ACC ATC
40 chromosome 19 aminoacidsequence
Numbers above DNA sequences indicate position of basc with rcspect to the 5'-end of the clone. Underlined bases indicate differences between the human sequences and the bovine cDNA. confirm the localization of the lcns MP19 gene to human chromosome 19. This is the second of the lens membrane intrinsic proteins to
be mapped to its specific human chromosome. MP26,the major lens intrinsic membrane protein has previously been mapped to human chromosome 12by Sparkes, d.(20). In the lens, the
423
Curr Eye Res Downloaded from informahealthcare.com by Flinders University of South Australia on 02/05/15 For personal use only.
Current Eye Research majority of the human crystallins have now been mapped, as well as two membrane proteins. To date, no correlation of the assigned genes for either crystallins or membrane proteins have been made with the various types of congenital cataract in man (2 1,22). Although it appears from the present gene assignment that the MP 19 gene is not associated with any of the presently known congential cataracts (these have been associated with human chromosomes 1,2, 14, 16, and X), it will be of interest to investigate other lens disorders and cataracts to determine if MP19 is associated in any way with the cataractous condition. ACKNOWLEDGEMENTS This work was supported in part by National Institutes of Health grants R01 EY08616, P30 EY06360, and C06 EY06307 and a Departmental Grant from Research to Prevent Blindness, Inc. CORRESPONDING AUTHOR Robert L. Church, Ph.D., Emory University School of Medicine, Department of Ophthalmology, 1327 Clifton Road, N.E.. Atlanta, Georgia 30322 REFERENCES 1 . Rao, G.N., Gutekunst, K.A. and Church, R.L. (1989) Bovine lens 23,2 1 and 19 kDa intrinsic membrane proteins have an identical amino-terminal amino acid 483-486. sequence. FEBS Lett. 2. Rao, G. N., Gutekunst, K.A. and Church, R.L. (1990) Bovine lens membrane uroteins: MP70. MP64. and MP38 are products of the same gene. Ophthal. Res. 2,166-172. 3. Bok, D., Dockstader, J. and Horwitz, J. (1 982) Immunocytochemical localization of the lens main intrinsic polypeptide (MIP 26) in communicating junctions. J. Cell Biol. =,213-220. 4. Gruijters, W.T.M., Kistler, J., Bullivant, S. and Goodenough, D.A. (1 987) Immunolocalization of MP70 in lens fiber 16-17 nm intercellular junctions. J. Cell Biol. M,565-572. 5 . Roy, D., Spector, A. and Famsworth, P.N. (1979) Human lens membrane: Comparison of the major intrinsic polypeptides from young and old lenses isolated by a new methodology. Exp. Eye Res. 28,353-358. 6. Gamer, W.H., Gamer, M.H. and Spector, A. (1981) Gamma-crystallin, a major cytoplasmic polypeptide disulfide linked to mebrane proteins in human cataract. Biochem. Biophys. Res. Commun. eS,439-447. 7. Kadama, T, and Takemoto, L. (1988) characterization of dusulfide-linked crystallins associated with human cataractous lens membranes. Invest. Ophthalmol. Vis. Sci. 29. 145-149. 8. Gutekunst, K.A., Rao, G.N. and Church, R.L. (1990) Molecular cloning and complete nucleotide sequence of the cDNA encoding a bovine lens intrinsic membrane protein (MP19). Curr. Eye Res. 9,955-961. 9. Louis, C.F., Johnson, R., Johnson, K. and Turnquist, J.
m,
424
(1 985) Characterization of the bovine lens plasma
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