Gene, 140 (1994) 257-259

0 1994 Elsevier Science B.V. All rights reserved. 0378-l 119/94/$07.00 257

GENE 07715

Nucleotide sequence of the bovine vimentin-encoding cDNA

(Amino acid sequence homology; intermediate filaments; bovine lens; cytoskeleton; epidermis)

John F. Hess, Jodi T. Casselman and Paul G. FitzGerald

Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, CA 95616, USA

Received by J. Piatigorsky: 5 April 1993; Revised/Accepted: 23 August/l 1 October 1993; Received at publishers: 18 November 1993

SUMMARY

Using reverse transcription-polymerase chain reaction (RT-PCR), we have amplified vimentin (Vim)-encoding se- quences from both total and polyadenylated bovine lens RNA. The amplified products were cloned and the nucleotide sequences determined. The amino-acid sequence of bovine vimentin shows 97.2, 96.8, 96.3 and 84.9% homology with reported aa sequences of human, mouse, hamster and chicken Vim, respectively.

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INTRODUCTION

The type-III intermediate filament (IF) protein vimen- tin (Vim) has been sequenced by translation of cDNA sequences isolated from human, hamster, mouse, chicken and other species (Ferrari et al., 1986; Wood et al., 1989; Quax-Jeuken et al., 1983; Zehner et al., 1987). In these species, the Vim-encoding DNA sequences were isolated from mesodermally derived tissues. However, in an excep- tion to the paradigm of mesodermal expression, Vim is also expressed in the ocular lens, an ectodermally de- rived tissue.

Vim is expressed in both the undifferentiated and differentiated cells of the ocular lens. As the lens cell differentiates, two additional cytoskeletal proteins are ex- pressed. These proteins, called filensin and phakosin, have recently been cloned and sequenced (Gounari et al., 1993; Hess et al., 1993). Both are related to the IF family, but are not clearly related to any of the existing classes

Correspondence to: P.G. FitzGerald, Department of Cell Biology and

Human Anatomy, School of Medicine, University of California, Davis,

CA 95616, USA. Tel. (1-916) 752-7130; Fax (1-916) 752-8520; e-mail:

JFHESS@UCDAVIS.EDU

Abbreviations: aa, amino acid(s); cDNA, DNA complementary to RNA;

IF, intermediate filament(s); nt, nucleotide(s); PMCC, plasma mem-

brane cytoskeletal complex; RT-PCR, reverse transcription-polymerase

chain reaction; Vim, vimentin; WIM, gene encoding VIM.

SSDI: 0378-1119(93)E0711-L

of IF proteins which have been defined. In order to ex- plore the assembly properties of these three IF proteins which co-exist in the same cell type, and to compare the degree of divergence between bovine Vim and Vim from other species, we have determined the nt sequence of bovine Km using RT-PCR of RNA isolated from bovine lenses.

EXPERIMENTAL AND DISCUSSION

(a) Determination of the nt sequence of bovine Vim

Western blotting of the plasma membrane- cytoskeleton complex (PMCC) proteins using Vim- specific antibodies confirms the presence of Vim in the PMCC. Locations of high sequence homology between human, hamster and mouse were chosen for the design and synthesis of oligos. Using these oligos, we amplified bovine I’im RNA sequences using RT-PCR (Frohman, 1990; Frohman et al., 1988). PCR products were cloned into pSP or pGEM standard cloning vectors and se- quenced following double-stranded protocols provided with the SequenaseTM and TaquenceTM DNA sequencing kits. The location and directionality of PCR primers, and the determined nt sequence of bovine T/im are presented in Fig. 1.

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1 61

121

181

241

301

361

421

481

541

601

661

721

181

841

901

961

1021

1081

1141

1201

1261

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1381

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lb21

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GGCAGCGCGCTGCGCCCCACCACCAGCCGCACCCTCTACACCTCGTCCCCGGGTGGCGTG

TACGCCACGCGCTCCTCGGCCGTGCGCCTGCGGAGCGGCGTGCCCGGCGTGCGGCTGCTG .

CAGGACTCGGTGGACTTCTCGTTGGCCGACGACGCCATCAACACCGAGTTCWGAACACCCGC

ACCAACGAGAAGGTGGAGCTGCAGGATGACCGC

GTGCGCTTCCTCGAGCAGCCTGCTGGCTGAGCTCGAGCAGCTCAAGGGC

CAGGGCAAGTCGCGCCTGGGGGAGCTCTACGAGGAGGAGATGCGAGAGCTGCGCCGGCAG ,

GTGGACCAGCTCACCAACGACAAAGCCCGCGTCGAGGTGGAGCGCGACAACCTGGCCGAQ

GACATCATGAGGCTCCGGGAGAAGTTGCAGGAGGAGATGCTTCAGAGAGAGGAAGCCGAG

AGCACTCTGCAGTCTTT04GACAG~TGTTGACAATGCCTCTTTGGCACGTCTTGACCTG .

GAGCGTAAAGTGGAATCCTTGCAAGAAGRGAAGA~TTT ,

GAAATCCAGGAGCTTCAGGCCCAGATTCAAGAACAGCATGTCC-TCGATAT~ATGTT

TCCAAGCCTGACCTCACGGCTGCCCTGCGTGATGTCCGTCAGCAGTATGAGAGCGTGGCC (

GCCAAGAACCTTCAGGAGGCTGAGGAATGGTACPAGTCCA

GCTGCTAACCGCAACAATGATGCCCTGCGCCTGCCAAGCCGC

CGGCAGGTGCAGACTCTTACCTGCGAGGTGGATGCGCTCATCTCTG

GAACGCCAGATGCGTGAAATGGAAGAGAACTTCTTCTCTGTGGAAGCTGCTAACTACCAA~

ACTATTGGCCGCCTGCAGGATGAGATTCAGGCCTG . .

C~GAATACCAAGACCTGCTG?&TGTCAAGATGGCGcTCGACATCGAGATCGcCACCTAC

AGGAAGCTGCTGGAAGGAGAGGAGAGCAGGATTTCTCTGCCTCTTCCAAWTTTTCTTCC

CTGAACCTGAGGGAARCCAATCTGGATTCACTCCCTCTGGTTGACACTCATTCAAJ@AGG

ACACTTCTGATTAAGACGGTGGAW,CCAGAGATGGKAGGTTATCAAC'.XP.kCTTCCCAG

CATCACGATGATCTGGAA@&AACTGcACATACTTCGTGCA GCARAATACTACCAGCW.

GAAGG~TCCATATCTTARAGAAACRGCTTTCAAGTGCCTTTCTGCAGTTT

TTTCAGGAGCGAAGATAGATTTMjAATAGGAATAAGCCCTAGTTCTTG~CC~CACCCG

TAATAGATTTAGAAWAA GTTTACAACATARTCTAGTTTACCGARGACGCCTTGTGCTAG

ARTACTTTTTAAAAAGTATTTTTGAATACCATTACCATT~CT~TTTTTTCCATC~GTATCT

GACCAACTTGTTTCTGCTTC#$@#$#CTTTGGAAAACTCTAAAA

Fig. 1. Nucleotide sequence of bovine lens Vim cDNA. The nt sequence

of both DNA strands was determined from plasmid clones using di-

deoxy chain termination methods. Start (ATG) and stop (TAA) codons,

and polyadenylation signal (AATAAA) are shaded. Primers used for

PCR and cloning are underlined, with directionality indicated by the

arrows. This sequence has been forwarded to GenBank and assigned

accession No. Ll3263.

(b) Vim sequence comparisons

The bovine Vim aa sequence is compared in Fig. 2 to

those determined previously in human, mouse, hamster

and chicken (Ferrari et al., 1989; Wood et al., 1989; Quax

et al., 1983; Zehner et al., 1987). When compared to con-

sensus sequence we find three conservative aa changes in

the N-terminal head domain. Within the central rod

domain (aa 103-410), we find three aa changes; a conser-

vative Met in place of Val”’ (within linker l-2), a conser-

vative Thr for Ser324 and a semi-conservative Ser for

Ala351 in rod domain 2B. The nt changes leading to the

aforementioned aa substitutions have been verified by

the independent amplification, cloning and sequencing of

cDNA (positions 46,.50,53,257,324 and 351) or genomic

DNA (positions 257,324 and 351). The net effect of these

changes on the molecule is unknown.

The N-terminal head domain is less stringently con-

served than the central rod domain and the substitutions

we observe are not within the region identified in vitro

Fig. 2. Comparison of bovine (Bo) Vim to human (Hu), mouse (MO),

hamster (Ha) and chicken (Ch) Vim. Those aa listed in bold type differ

from the Vim consensus sequence.

as important for lateral packing and alignment of the

subunits (Herrmann et al., 1992). The substitutions iden-

tified in the rod domain are either conservative (aa and

aa324) or semi-conservative (aa351). The substitution at

aa does not introduce or remove a charged aa and is

not one of the A or D positions of the heptad repeat

pattern usually occupied by non-polar aa. For these

reasons, no structural changes are likely, and have not

been observed by examination of intermediate filaments

in the electron microscope (Maisel, 1985; P.G.F., unpub-

lished data).

ACKNOWLEDGEMENTS

Research supported by funding from National Institute

of Health grant No. EY08747 to P.G.F.

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259

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