Connexin mutations in X-linked Charcot-Marie-Tooth disease

Science

Volumn 262, Issue 5142, 1993, Pages 2039-2042

  Bergoffen, J ^a,b   Scherer, S S ^b   Wang, S ^b   Oronzi Scott, M ^b   Bone, L J ^b   Paul, D L ^c   Chen, K ^b   Lensch, M W ^b   Chance, P F ^b   Fischbeck, K H ^b  

^a KAISER PERMANENTE MEDICAL CENTER   (United States)

^b UNIVERSITY OF PENNSYLVANIA   (United States)

^c HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GAP JUNCTION PROTEIN;

ARTICLE; CELL JUNCTION; CLINICAL ARTICLE; CONTROLLED STUDY; FEMALE; HEREDITARY MOTOR SENSORY NEUROPATHY; HUMAN; IMMUNOHISTOCHEMISTRY; MUTATION; NORTHERN BLOTTING; PRIORITY JOURNAL; PROTEIN DEFECT; X CHROMOSOME LINKED DISORDER;

EID: 0027772413     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.8266101     Document Type: Article

References (35)

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28. The technique for Northern blot analysis was as described [B. Friedman et al., Neuron 9, 295 (1992)]. Total RNA was isolated from the indicated rat tissues as described [J. M. Chirgwin, A. E. Pryzbyla, R. J. MacDonald, R. J. Rutter, Biochemistry 18, 5294 (1979)]. The RNA was separated on a 1% agarose gel, transferred to a nylon membrane overnight in 6x saline sodium citrate (SSC), and hybridized overnight with a radiolabeled complementary DNA probe for rat Cx32 (7). The blot was washed at a final stringency of 0.2x SSC for 30 min at 65°C and exposed to x-ray film with an intensifying screen for 5 days at -80°C. Approximately equal loading of samples in each lane was assured by comparison of the intensity of ethidium-stained ribosomal RNA bands in the gel and by rehybridization of the membrane with a complementary DNA probe for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase.
29. The technique for Northern blot analysis was as described [B. Friedman et al., Neuron 9, 295 (1992)]. Total RNA was isolated from the indicated rat tissues as described [J. M. Chirgwin, A. E. Pryzbyla, R. J. MacDonald, R. J. Rutter, Biochemistry 18, 5294 (1979)]. The RNA was separated on a 1% agarose gel, transferred to a nylon membrane overnight in 6x saline sodium citrate (SSC), and hybridized overnight with a radiolabeled complementary DNA probe for rat Cx32 (7). The blot was washed at a final stringency of 0.2x SSC for 30 min at 65°C and exposed to x-ray film with an intensifying screen for 5 days at -80°C. Approximately equal loading of samples in each lane was assured by comparison of the intensity of ethidium-stained ribosomal RNA bands in the gel and by rehybridization of the membrane with a complementary DNA probe for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase.
30. Blood was obtained from CMTX patients by informed consent, and genomic DNA was isolated from venous blood by standard methods (4). The connexin32 coding region was amplified with PCR with various primer sets spanning this region. The segment including bases 54 to 359 (13) was amplified with the primer set 5′-TGAGGCAGGATGAAC-TGGACAGGT-3′ (bases 54 to 77) and 5′-TTGCTG-GTGAGCCACGTGCATGGC-3′ (bases 336 to 359), and the segment including bases 273 to 938 was amplified with the primer set 5′-ATCTCCCATGT-GCGGCTGTGGTCC-3′ (bases 273 to 296) and 5′-TGGCAGGTTGCCTGGTATGT-3′ (bases 919 to 938). Twelve picomoles of each primer set was used in a reaction volume of 100 μl with 100 to 200 ng of genomic DNA as a template. PCR conditions for the first primer set were 94°C for 7 min; 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s (35 cycles); and 72°C for 10 min. Conditions for the second primer set were 94°C for 5 min; 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min (35 cycles); and 72°C for 10 min. Amplified products were purified with the Gene Clean protocol (BIO 101) and subsequently used as templates for sequencing at 20 to 50 ng per reaction. The primers listed above were used for sequencing; in addition, the following primers were used for bases 273 to 704 and 635 to 933, respectively; 5′-GATGATGAGGTACACCACCT-3′ (bases 685 to 704) and 5′-CGTCTTCATGCTAGCTGCCTC-TGG-3′ (bases 635 to 658). The sequencing reactions were set up according to manufacturer's recommendations with the Sequenase Version 2.0 kit (U.S. Biomedicals) with minor modifications (10 pmol of primer per reaction; 50°C termination for 5 min).
31. 12 → Ser is given by G12S.
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34. Adult rat sciatic nerve was fixed in 1.6% formaldehyde in Hank's buffered salts (Gibco) for 1 hour at room temperature. The tissue was then transferred to tris-buffered saline (TBS) and incubated for 2 hours at 4°C, followed by incubation in 0.5 M sucrose overnight for cryoprotection. Samples were quick-frozen in OCT (Miles Scientific, Elkhart, IN) with liquid freon, and 10- to 12-μm cryosections were collected. The sections were mounted on slides, air dried, then incubated in acetone for 5 min at room temperature. After rehydration in TBS for 5 min, the sections were blocked for 15 min in 2% fish skin gelatin (Sigma), 1% normal goat serum, and 0.25% Triton X-100 in TBS (blocking solution), then incubated with a 1/1000 dilution of Cx32 antiserum (anti-98/124) [D. A. Goodenough, D. L. Paul, L. Jesiatis, J. Cell Biol. 107, 1817 (1988)] in blocking solution for 1 hour at room temperature. The sections were washed twice in 50 ml of TBS with 0.25% Triton X-100 and once with TBS, then incubated with 1/500 rhodamine-conjugated goat antibody to rabbit (Pierce) in blocking buffer. The sections were washed and mounted for immunofluorescence microscopy as described (12).
35. We thank the families studied for their cooperation; J. P. Fryns, M. Rozear, M. Pericak-Vance, and J. Stajich for help with sample collection; C. Litrenta and Y.-T. Yu for technical assistance; I. Corcos for providing the Cx32 cDNA probe; C. Lo, J. Kamholz, J. Garbern, and H. Paulson for helpful discussions; and V. Valmiki for help with figure preparation. Supported with grants from the Muscular Dystrophy Association, the March of Dimes Birth Defects Foundation, and NIH (NS08075, NS01565, GM37751, and NS30804). J.B. was supported by a fellowship from The Charles A. Dana Foundation.