Identification of a novel myosin heavy chain gene expressed in the rat larynx

BiBBochi~ie~a et Biophysica A~ta

ELSEVIER

Biochimica et Biophysica Acta 1306 (1996) 153 - 159

Identification of a novel myosin heavy chain gene expressed in the rat larynx Albert L. Merati

a

Sue C. Bodine b Thecla Bennett a Hak-Hyun Jung Allen F. Ryan a,*

a

Hiroshi Furuta

a

a Department t~fSurgery/Otolarygology, UCSD Medical School and VA Medical Center, La Jolla, CA 92093, USA b Department of Orthopedics, UCSD Medical School and VA Medical Center, La Jolla, CA 92093, USA Received 7 March 1995; accepted 6 December 1995

Abstract Based on reactivity to antibodies against known myosin heavy chains, expression of a novel fast myosin heavy chain (MHC) gene was suspected in the thyroarytenoid (TA) muscle of the rat larynx. The 3' ends of MHC transcripts in the TA were amplified by RT-PCR using a primer to a highly conserved MHC sequence and to the poly(A) tail. The resultant products were cloned and fourteen PCR products were screened by dot-blotting with oligonucleotides specific for known skeletal muscle MHC genes. A clone that reacted weakly to the 2B oligo was sequenced and found to encode a novel fast MHC transcript, termed 2L, that appears to represent an eighth vertebrate skeletal muscle MHC gene. F|y homology analysis, the 2L sequence is most similar to the extraocular MHC, suggesting a possible evolutionary relationship between MHCs associated with the branchial arches. Keywords: Muscle protein; Larynx; Myosin heavy chain gene; Myosin evolution; (Rat)

I. Introduction Mammalian skeletal muscles have a range of structural and functional properties which are related, in part, to the type of myofibrillar myosin expressed. The myosin heavy chain gene family encodes raajor isoforms which are widely distributed in most mammalian skeletal muscles, and minor isoforms which are restricted to specialized muscles. To date, seven MHC genes have been identified through expression in rat skeletal muscles. Two of these genes encode developmental isoforms: embryonic [1] and neonatal [2]. The slow isoform is equivalent to the j3-cardiac MHC [3,4]. Three fast MHC genes have been isolated from limb muscle transcripts: 2A [5,6], 2X [7,8], and 2B [5,6]. A seventh MHC gene is expressed in extraocular muscles, but not in limb muscles [9]. Other MHC genes may exist. A 'superfast' MHC isoform has been identified based on physiology and histochemistry in the masseter muscle of primates and cm'nivores [10], but this myosin has not been characterized at the molecular level.

* Corresponding author. Fax: + 1 (619) 5345319. 0167-4781/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 (95)00237-15

MHC isoforms differ in their functional properties. The speed of contraction of muscle fibers depends, in part, on the MHC isoform expressed [11,12]. MHC isoforms also show different substrate affinities [13] and energetic efficiencies [14]. The unique functional characteristics of the specialized muscles of the head and neck, which include very rapid contraction, suggest that MHC isoforms expressed in these muscles may well be different from those in the limb musculature. This is supported by the existence of tissue-specific isoforms in the extraocular muscles of the rat [9] and in the cat masseter [10], as mentioned above. Additional MHC genes may also be expressed in other muscles of the head and neck. The intrinsic laryngeal muscles are non-somitic muscles that, like the masseter, are derived from the branchial arches. The larynx functions in vocalization and in the regulation of ventilation, making it a highly specialized and critical structure. The thyroarytenoid (TA) muscle is the principal adductor of the vocal cords. It is activated in laryngeal reflexes to rapidly seal and protect the airway, and also operates in phonation [15]. While most head and neck muscles are characterized by rapid contraction times compared to the limb muscles, the TA is particularly fast [15-17].

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Although the MHC compositions of muscles of the mammalian hindlimb have been well characterized, relatively little is known about the muscles in the head and neck, including laryngeal muscles. The fiber type characterization which has been performed in the larynx of several species has been limited to the myofibrillar adenosine triphosphatase histochemical reaction [18-21]. While this work has clearly identified a predominance of fast MHC in the TA, the method is unable to distinguish between the various fast MHC isoforms. In the course of an immunohistochemical study of MHC isoforms in laryngeal muscles, evidence for a unique MHC isoform in the TA was encountered. The purpose of the present study was to isolate and characterize the mRNA transcript encoding this isoform.

2. Materials and methods

2.1. Subjects Young adult (60-90 days old) male Harlan SpragueDawley rats were used as subjects in all experiments. All procedures were reviewed and approved by the local IRB and conformed to NIH guidelines for the use of animal subjects.

2.2. Immunocytochemistry Rats were anesthetized with rodent cocktail (ketamine 50 mg/kg, rompun 5 m g / k g and acepromazine 1 mg/kg). The intact larynx was isolated and frozen immediately in isopentane cooled by liquid nitrogen. The thyroarytenoid (TA) muscle was identified in serial cross-sections following immunohistochemical staining with monoclonal antibodies against the MHC. Serial cross-sections were incubated overnight at 25°C with primary antibodies (BA-F8, BF-13, BF-35 and SC-71) generously provided by S. Schiaffino (Padova, Italy). Sections incubated without primary antibody were used as controls to visualize nonspecific labeling. A Vectastain ABC TM kit (Vector Labs, Burlingame, CA, USA) was used to amplify the antigenantibody complex, which in turn, was visualized by treatment with a DAB peroxidase reaction. Immunohistochemical labeling was compared to standard histochemical staining for myofibrillar ATPase after acid (pH 4.35) or alkaline (pH 10.0) preincubation [22].

Table 1 Oligonucleotide sequences used for cloning and screening Modified poly(T) primer Consensus MHC sense primer Specific 3' antisense primers: Embryonic MHC Neonatal MHC Slow MHC 2A MHC 2B MHC 2X MHC Extraocular MHC 2L MHC

GCGGATCCTTTTTTT'UI~ItVITT AGAAGGCCAARAARGCCAT ATGTGGAAAGGGGTTACGT AGTCAGCAGTGGGAGAAAAG TTTCTGCCTGAAGGTGCTGT TTACAATAGGATTAAATAGAA TTGATATACAGGACAGTGACA TTTTTTATCTCCCAAAGTCG ATTCCAGTCTCCTCTGCTCT GAGGCCAGAAGATGGAAGAA

2.3. Molecular biology 1-2 mg of TA muscle tissue was harvested from anesthetized rats and immediately frozen on dry ice. Upon thawing, the tissue was homogenized, mRNA was harvested using poly(T) on Dynabeads (1 rag, Dynal) and phenol extracted according to the manufacturer's recommended protocol, mRNA was not measured, but due to the capacity of the Dynabeads could not have exceeded 2 /zg. A reverse transcription-polymerase chain reaction (RTPCR) procedure [6] was then performed on the entire sample of mRNA to amplify the 3' ends of all MHC transcripts. RT-PCR was performed with the cDNA Cycle Kit (Invitrogen) except for the use of a poly(T) primer modified by the addition of a BamHI site and enough additional bases to raise the melt temperature (Tm) to 60°C on the 5' end (Table 1). The upstream primer was a degenerate oligonucleotide from a highly conserved region occurring from 620-660 bases from the 3' end of all known rat MHC genes, as described previously [6], and having an average Tm of 60°C (Table 1). After denaturation at 94°C for 2 min under oil, Taq polymerase (2.5 units) was added, and thirty cycles of PCR were performed using the following protocol: 94°C for 1 rain, 58°C for 2 min, 72°C for 1 min. The resulting mixed PCR product was cloned into the pCRII vector (Invitrogen) using the manufacturer's recommended protocol. Fourteen clones were isolated, minipreps were performed, and the inserts were sized on 1% agarose gels to confirm the expected 620-660 bp size range. Clones were screened by dot blot [23] using 32P-end-labeled 20-mer oligonucleotides complementary to unique sequences within the 3' UT regions of rat slow, 2A, 2X, 2B,

Fig. 1. Immunocytochemical evidence for a unique myosin in the rat thyroarytenoid (TA). Serial coronal sections through the larynx were stained immunohistochemically with an antibody (BF-13) which recognizes all fast MHC isoforms (2A, 2B and 2X) or an antibody (BF-35) that recognizes all MHC isoforms except 2X. The bilateral TA muscles adjacent to the vocal folds are shown. No fibers in the TA labeled with an antibody specific for slow MHC (not shown). All but a few fibers of the largest region of the TA stained weakly with antibody BF-13 (panels A and B). Those few that were positive for BF-13 (arrow, panel B), were negative for antibody BF-35 (arrow, panel C), indicating that they are 2X fibers. However, most fibers in this region stained positively for BF-35, indicating the presence of a MHC isoform that does not fit into the known classification scheme. (Original magnifications: Panel A, 12.8 X ; Panels B and C, 40 X .)

A.L. Merati et al. / Biochimica et Biophysica Acta 1306 (1996) 153-159

extraocular, embryonic or neonatal MHC (Table 1). Each dot contained 100 ng of purified plasmid DNA, and was cross-linked to a nitrocellulose membrane by UV irradia-

155

tion (Stratalinker, Stratagene). Blots were blocked with denatured salmon sperm DNA in Church's buffer, hybridized at 40°C overnight with 5. l 0 6 cts/ml of probe,

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A.L. Merati et al. / Biochimica et Biophysica Acta 1306 (1996) 153-159

and washed at low stringency (40°C, 30 min, 2 × SSC + 0.05% SDS) followed by a first high-stringency wash (42°C, 30 min, 2 × SSC + 0.05% SDS), and a final highstringency wash (50-65°C, depending upon probe melt temperature, 60 min, 5 × SSC + 0.1% SDS). Clones of interest were sequenced using the double-stranded dideoxy method [24]. MHC cDNA and predicted protein sequences were aligned using a modification of the procedures described by Smith et al. [25], as implemented in the GeneWorks software package (Intelligenetics). The alignment strategy was configured to make the cost of opening a gap half the cost of lengthening a gap, which has been shown to be highly effective when comparing sequences with substantial homology. Since D N A alignment is nucleotide number sensitive, the 5' ends of all D N A sequences were trimmed to match the shortest sequence, that of extraocular MHC [9]. The sequences included the 3' UT regions to approximately 20 bases past the polyadenylation signal. A separate alignment was performed on the 3' UT regions alone. Total R N A was isolated from fresh samples of rat liver, heart muscle, masseter, hindlimb muscle and strap muscles from the neck by a single step, guanidium thiocyanate-phenol-chloroform extraction [26] for Northern blotting [27]. A total of 5 /xg of each RNA was size separated by electrophoresis on an agarose/formaldehyde gel and transferred to a nylon membrane. 32P-labeled probe was synthesized by random priming (Prime-It II kit, Stratagene) using the 3' 270 bp, which showed the least homology to other published MHCs, of a unique PCR product isolated from the TA. The membrane was blocked with salmon sperm D N A and hybridized at 68°C overnight. The blot was washed four times for one hour each at 68°C (2X, 40 mM NaPO 4, 5% SDS, 0.5% BSA, 1 mM EDTA; 2X, 40 mM NaPO 4, 2% SDS, 1 mM EDTA). The blot was exposed to X-ray film for 9 days.

3. Results 3,1. I m m u n o c y t o c h e m i s t r y

!

2

3

4

5

6

7

8

9

10

11

12

13

14

2X

Emb

2A

2B

Slow

Fig. 2. MHC clones from the TA hybridized to a 2B probe. The blot consisted of 14 clones from a MHC PCR product amplified from TA cDNAs, as well as control cDNA fragments from the corresponding region of five known MHCs: 2X, embryonic (Emb), 2A, 2B and /3 cardaic (Slow). The blot was hybridized using a 32P-labeled oligonucleotide corresponding to a 20-base segment of the 3' UTR of 2B MHC mRNA. Most clones bound strongly to the 2B probe. Three clones (clones 1, 5 and 8) showed no or very weak labeling. All other MHC probes tested failed to hybridize to any of the 14 TA clones, although the 2X, embryonic, 2A, 2B and slow probes bound specifically to their control DNAs. fourteen clones produced from this product showed no reactivity to oligonucleotide probes against slow, 2A, 2X, extraocular, embryonic or neonatal MHC. Eleven clones hybridized strongly to the 2B oligonucleotide, while three clones showed no or very weak hybridization (Fig, 2). The latter three clones were selected for sequencing. One clone was found to be completely homologous to 2B, but with deletion of a short region in the 3' UTR, including the sequence of the probe, presumably due to a cloning artifact. One of the other two clones was partially sequenced, and showed only two base pairs different from A A G G C C ATC A C T G A C O C C G C C A T G ATG G C G G A G G A G CTG A A G A A G G A G C A G G A C Lys Ala He Tb/ Asp Ala Ala Met Met A h Glu Glu Leu Lys Lys Glu Gin Asp

54 I$

A C C A G C G C C C A T CTG G A G C G G A T G A A G A A G A A C CTO G A G C A G A C G G T G A A G G A C Thr Ser Ala Kis Lea Glu Arg Met Lys Lys A ~ Leu Glu Gin Thr Val Lys Asp

i06 36

CTG CAG CAC CGT CTN GAC GAG GC~ GAG CAG CTG GCG CTG AAG GGC CTG AAG GGC Leu Gin His Arg Lee Asp GIu Ala GIn Gin Leu Ala Lee Ly$ Gly Lea Lys Gly

162 54

GGC AAG AAO CAG ATC CAG AAA CTG GAG GCC AGAGTG CCK3GAG ~ GAA AGC GAG 216 Gly Lys Lys Gin lie Gkt Lys Leu Glu Ala Arg Val Arg Glu Leu Olu Sex Glu 72

None of the fibers in the TA reacted with antibodies that stain slow MHC. A population of fast fibers could not be classified based on known staining characteristics of anti-MHC antibodies. In particular, a large subset of fibers in the TA stained very lightly with an anti-fast antibody (BF-13) which normally labels all fast MHCs in limb muscles (2A, 2X and 2B) strongly (Fig. 1). It was this unusual staining pattern that led us to suspect that a unique MHC isoform existed in the TA. 3.2. M o l e c u l a r biology

RT-PCR of m R N A from the TA muscle yielded, as expected, PCR products of approximately 640 bp. The

C'I~ GAT GCA GAG CAG AAG AGAGGA GCT OAA GeT CTG AAG GGG GGC CAC AAA TAT Lee Asp Ala nla Gin Lys Arg Gly Ala Olu Ala Lea Lys Gly Gly His Lys Tyr

270 90

GAG CGC AAA OTC AAA GAG ATG ACT TAC CAG GCC GAG GAG GAC CGC AAG AAC ATC Glu Arg Lys Val Lys Ola Met Thr Tyr Gin Ala Glu GIu Asp Arg Lys ASh ne

324 108

CTC CGA CTC CAG GAC CTG GTA OAC AAG CTG CAG GCC AAA GTG AAG TCC TAC AAG Lea Arg Leu Gin Asp Lee Val Asp Lys Leu Gin Ala Lys Val Lys Sex Tyr Lys

378 126

AGG CAG GCA GAA GAG GCT GAG GAG CAA GCC AAC ACA CAG CTA TCC AGG TGC CGG Arg Gin Ala Glu GIu Ala Glu Glu Gin Ala Ash Thr Gin Leu Set Arg Cys Arg

432 144

AGG GTC CAG CAT OAA eTA GAG GAG GeT GAG GAG AGG GCA GAT ATC GCT GAG TCT Arg Val Gin His GIu Leu Glu Glu Ala Glu Glu Arg Ala Asp Ile Ala GIu Set

486 162

CAA GTC AAC AAG CTA AGG OCC AAG AGC CGT OAT OTG GGA GGC CAG AAG ATG GAA Gin Val A~n Lys Lea Arg Ala Lys S~r Arg Asp Val Gly Gly Gin Lys Met Glu

540 180

GAATGA 8 g ~ ¢ c c g a g g c ~ g l I g ~ a t S g g c c a ~ ¢ c a g g a S ~ t g c a L ~ t c a a a a g a n Glu

632 181

Fig. 3. Partial cDNA sequence and deduced amino acid sequence of the 3' end of rat 2L MHC (clone 8 from Fig. 2). The sequence corresponds to the carboxy-terminusof the linear tail of the MHC molecule. The putative polyadenylation signal is underlined.

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Ma

Ne

Lm

He

Lv

7.46 4.40

1.35

Fig. 4. Northern blot showing hybridizationof an approx. 6 kb message in total RNA isolated from rat masseter (Ma) and hindlimb (Li) but not strap muscles from the neck (Ne), heart muscle (He) or liver (Lv).

2B in the sequenced region. The third clone was completely sequenced and proved to have a DNA and predicted amino acid sequence: quite different from 2B, or any other known MHC gene. The clone was 632 bases long, encoding 181 amino acids from the carboxy-terminus of a predicted protein product, and with an 89 base 3' UTR prior to the poly(A) tail (Fig. 3). While the 5' half of the sequence was highly homologous to those of other MHC genes, the 3' half showed significant sequence deviation. This unique MHC was named ' 2 L ' . An oligonucleotide was synthesized based on the sequence of 2L and hybridized against the original dot blot of fourteen TA MHC clones. The probe reacted strongly with the original 2L clone, and failed to hybridize to any other clone. A probe synthesized from the 3' 270 bp of the 2L clone, which showed the least homology to other MHCs, hybridized to a 6 kb message in a Northern blot of total RNA from masseter, and faintly to a similar message in hindlimb

KAKKAITDAA ~£~EELKKEQ not available F~/~EAITDAA ~RkEEEE4~EQ F~tK~AITDAA ~ M A F ~ Q KAKKAITDAA MMAEELKKEQ KAK~AITDAA MMAEELKXEQ FJ~KAITDAA MMAZEL~XEQ KAKKAITDAA MMAEELKKEQ KAKKAITDAA ~4AEELKKEQ

DTSAH~ DTSAHLER~ DTSAHLERMK DTSAHLER~ DTSAHLEPh~ DTSAHLER~A~ DTSAHLER~

2L EO 2A 2B 2X Neo ~bry Slow G Card

ALKGGKKQIQ KLEARVRELE not available ALKGGKKQIQ KLEARVRELE ALKGG~K~IQ KLEARVRELE ALKGGKXQIQ KLEARVRELE ALKGGKKQIQ KLEARVRELE ALKGGKKQIQ KLETRIRELE ALKGGKKQLQ KLEARVRELE ALKGGKKQLQ KSEARVRELE

GEVESE0~RN NEVENEQKRN GEVENEQKRN GE~rENEQKRN FELEGEQKRN MELEAEQKRN NELEAEQKRN

VEAVKGLRKH IEAVKGLRKH VEAIKGLRKH AEAVKGLRKH TESVKGLRKY AESVKG~RRKS AESVKG~RRKS

ERRVKELTYQ ERRVK~LTYQ ERRVKELTYQ ERRVKELTYQ ERRVKELTYQ ERRIKELTYQ ERRIKELTYQ

100 100 100 10O 100 100 100 100 100

2L EO 2A 2B 2X Neo E4~bry Slow G Card

AEEDRKNILR LQDLVDKLQA not available TEEDR~WILR LQDLVDKLQA TEEDRKNVLE LQHLVDKLQT TEEDRENVLR LQDLVDKLQS TEEDRm~VLR LQDLVDKLQA SEEDFA~VLR LQDLVDKLQV TEEDRENLLR LQDLVDKLOL TEEDKENLVR LQDLVDKLQL

KVKSYKRQAE EAEEQANTQ5 AEEQANTQL KVKSYKRQAE EAEEQSNTNL KVKAYKRQAE EAEEQSNVNL KVKAYKRQAE EAEEXSNVNL KVKSYKRQAB EAEEQSNVNL KVKSYKRQAE EADEQANVHL KVKAYKRQAE EAEEQANTNL KVKAyKP~2AE EAEEOANTNL

SRCRRVQEE5 SKFRKVQEEL SKFRKLQHEL AKFRKIQEEL AKFRKIPARV AKFRKLQEEL TKFRKAQHEL S~RKVQHEL SKFRKVQHEL

150 150 150 150 150 150 150 150 150

2L EO 2A 2B 2x Neo Embry Slow Card

EEAEERADIA EEAEERADIA EEAEERADIA EEAEERADIA EEAEERADIA EEAEERADIA EEAEERADIA DEAEERADIA DEAEERADIA

SRDVGGQ--K - - - ~ SRD---K . . . . . . NEE SREVHTK--V I--SEE SREVETK--V I--SEE SREVHTK--I I--SEE SREVHTK--I ---SAE TRDFTSSRMV VEESEE SRDIGAKG-L ---NEE SRDIGAKQKM H--DEE

ESQVNKLRAK ESQ~WKLRFK ESQVNKLRVK ESQVNKLRVK ESQVNKLRVK ESOVNKLRVK ESQVNKLRAK ESQVNKLRAK ESQVNKLRAK

DTSAHLERMK KNLEQTVKDL QHRLDEAEQL

50 50 50 50 50 50 50 50 50

2L EO 2A 2B 2X Neo e~Dry Slow Card

~4EQT~L T~VIDL KNLEQTV~DL ENJ~TVKDL ~ L HN~QTIEDL ~QTIKDL

QLRLDEAEQL QHRLDEAEQL QHRLDEAEQL QHRLDEAEQL QHRLDEAEQL QHRLDEAEQI QERLDEAEQI

SELDAEQKRG AEALKGGHKY ERKVKEMTYQ

181 176 (presR~ed) 182 182 182 181 186 182 184

Fig. 5. Protein alignment of 2L MHC with other MHCs. Alignment of 181-186 deduced amino acid residues from the carboxy-terminiof rat 2A, 2B, 2X, slow, c~ cardiac, embryonicand neonatalMHC sequencesis shown. Only the terminal 46 residues of the extraocular MHC are available.

muscle RNA, but not to RNA from strap muscles in the neck, heart muscle, or liver (Fig. 4). This message length is comparable to that of other MHC mRNAs [ 1,3,4]. The homologies of the novel sequence to other MHC genes are presented in Table 2. The 2L MHC isoform showed sequence homology to other rat MHC transcripts ranging from 52 to 76%, with the greatest similarity being to extraocular myosin. The sequence homology between

Table 2 Percent homologyof the terminal[340 bp, and the 3' untranslatedregion (in parentheses), of the rat 2L MHC sequence with the correspondingregions of seven other rat skeletal muscle MHC genes 2L 2A 2B 2X EO Neonatal Embry Slow ot Card

2L

2A

2B

2X

100 (100) 54 (28) 58 (34) 52 (27) 76 (60) 56 (11) 52 (12) 53 (27) 58 (28)

100 (100) 70 (45) 75 (57) 56 (33) 75 (58) 63 (29) 62 (28) 58 (28)

100 (100) 78 (58) 55 (36) 65 (33) 56 (38) 66 (25) 61 (25)

100 (100) 53 (6) 66 (38) 51 (16) 64 (24) 57 (24)

EO

Neo

Emb

Slow

ot Card

100 (100) 60 (23) 60 (6) 56 (6)

100 (100) 46 (23) 58 (23)

100 (lOO) 62 (32)

lOO (100)

100

(100) 55 (26) 56 (32) 56 (31) 62 (31)

158

A.L. Merati et al. / Biochimica et Biophysica Acta 1306 (1996) 153-159

the 3' UTR of 2L and extraocular MHC is particularly striking. At 60%, it was far higher than the homology with the 3' UTRs of any other MHC, for both 2L (11-34%) and extraocular (6-38%) MHC. A similar close relationship was observed between the 3' UTRs of the 2X and 2B (58%) and the 2A and neonatal (58%) MHCs. Fig. 5 shows an alignment of the predicted amino acid translation of the 2L sequence with those of seven other skeletal muscle MHCs. From the alignment it is clear that for many of the differences between 2L and other MHC DNA sequences, the amino acid sequence is conserved. The major differences in amino acid sequence are in the region from 70-100 amino acids from the start of each MHC fragment, involving substitutions of amino acids with similar characteristics, and in the final ten amino acids. In particular, the seven-residue repeat structure characteristic of a-helical coiled-coil molecules and organized into 28-residue repeats in MHC tail regions [1,28], is preserved in the 2L MHC.

4. Discussion The sequence of the 2L clone appears to encode a novel MHC. One possible origin of this sequence is through splice variation of a known MHC gene. The exon structure of MHCs is highly conserved across several isoforms and species [13], including mammalian and chicken genes [29]. It has been completely characterized for rat embryonic myosin [1], and partly characterized for rat extraocular myosin [9]. If as seems likely the exon structure of the putative 2L gene is similar to that of other mammalian MHC genes, the cloned region represents a portion of exon 36 as well as exons 37-41. Alternative exons have not been identified in genomic clones including this region [1,9], and splice variation of these exons has not been reported previously in MHC mRNAs [13]. A region of sequence variation between the 2L and extraocular cDNA sequences does span the boundary between exons 40 and 41. However, all other sequence variation is independent of intron/exon boundaries. It therefore seems unlikely that the 2L transcript originated by alternative splicing of a known gene. Rather, the transcript appears to have originated from a novel MHC gene. Hybridization of the dot-blot of fourteen MHC PCR products from the TA suggests that the predominant isoform in this muscle is 2B, with 2L representing a less abundant isoform. However the sample is small, and the relative numbers of clones might be the result of differences in PCR or cloning efficiency. The failure of immunohistochemistry to identify 2B in the TA could be the result of co-expression of 2B and 2L in the same fibers, since co-expression of MHC isoforms can alter immunolabeling of fibers (S. Bodine-Fowler, unpublished observation). Co-expression of fast MHC isoforms has been observed in fibers of hindlimb muscles [30].

It is possible that 2L is related to the so-called 'superfast' isoform detected in the cat masseter [10]. This isoform appears to be widely expressed in branchial arch muscles of carnivores and primates [31], and the contraction time of the masseter [32] is comparable to that of the TA [15-17]. The hybridization of 2L probe to a 6 kb message in RNA from the masseter supports this possibility. However, it should be noted that the superfast isoform has not been detected in the masseter or other branchial arch muscles of rodents [31,33]. The close homology between the available segments of the 2L and extraocular MHC mRNAs agrees with a recent report that antibodies raised against extraocular MHC react with fibers in the TA [34], and suggests that these two MHCs may have diverged from an ancestral MHC gene associated with the branchial arches relatively recently in evolution. The homology observed between the 3' UTRs of the 2L and extraocular MHCs was particularly striking. The 3' UTRs of many MHC genes contain a conserved 40 bp sequence of unknown function [35]. However the homology between 2L and extraocular MHC 3' UTRs covered a much broader area. Shoemaker et al. [8] have suggested that short ( ~ 10 bp) sequences within the 3' UTR of MHC molecules may be related to mRNA trafficking. If true, this might also contribute to the similarity between the 3' UTRs of 2L and extraocular myosin. It is possible that the existence of a 2L MHC isoform is related to the functional characteristics of the TA muscle, including the rapid contraction time dictated by the protection of the airway in laryngeal reflexes. MHC isoforms differ in their contractile behavior, although the myosin light chain with which the MHC is paired also has a significant influence on contractile properties [30]. Since the amino acid substitutions observed in the tail region of the 2L MHC were conservative, it is unlikely that the characteristics determined by this region, such as filament assembly [36], would be different in this isoform. However, other regions of the 2L molecule may show divergences that have functional consequences. Additional 2L sequence will be required to address this issue. The existence of a separate gene to encode 2L MHC may be related to other aspects of MHC gene expression besides the structure of the expressed protein. For example, at least some head and neck muscles appear to be subject to unique developmental regulation compared to limb muscles [32]. The significance of this novel MHC gene will be the subject of future investigations.

Acknowledgements This work was supported by grants DC00129, DC00028 and AR35192 from the NIH, NAG2-714 from NASA, and by the Research Service of the VA.

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[19]

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[24] [25] [26] [27]

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