Molecular and Biochemical Parasitology, 56 (1992) 345-348 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
345
MOLBIO 01868
Short C o m m u n i c a t i o n
Cloning of a cDNA encoding
-tubulin from Haemonchus contortus
R o n a l d D. Klein a, Susan C. N u l f b, Susan J. A l e x a n d e r - B o w m a n b, C a r o l y n B. M a i n o n e a, Christal A. W i n t e r r o w d b and T i m o t h y G. G e a r y aMolecular Biology Research and bAnimal Health Therapeutics Research, Upjohn Laboratories, The Upjohn Company, Kalamazoo, MI, USA (Received 12 June 1992; accepted 8 September 1992)
Key words: ct-tubulin cDNA; Haemonchus contortus; Nematode
Microtubules, which are polymers of dimers of ct- and fl-tubulin, have diverse roles in cellular morphology, axonal transport and cell division [1]. Most eukaryotic organisms express multiple, distinct isotypes of ~- and fltubulin, though specific functional differences among these isotypes have generally not been discerned (see ref. 2 for review). A particularly intriguing aspect of tubulin in nematodes is that the benzimidazole class of anthelmintics acts to destabilize microtubules by disrupting the ~-fl dimer (see refs. 3 and 4). This interaction is selective for nematode tubulin [5,6], which explains the chemotherapeutic utility of these drugs. The molecular basis for this selective toxicity is not yet clear. In order to better understand the molecular pharmacology of these important drugs, we have cloned and characterized 3 fl-tubulin cDNAs from H a e m o n c h u s contortus, a parasite of the ruminant abomasum [7]. In this report, we describe an ~-tubulin cDNA cloned from the Correspondence address: R.D. Klein, 7242/267/5, The Upjohn Company, Kalamazoo, MI 49007, USA. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankT M data base with the accession number L-02108. Abbreviations: SSC, standard saline citrate; ORF, open reading frame.
same organism. An immature adult H. contortus cDNA library in the vector 2ZAPII [8] was plated at a density of 5 x 104 pfu per plate and screened with a mouse ~4 tubulin cDNA (ref. 9; a gift from N. Cowan, New York University) at 65°C in 0.5 x SSC. Hybridizing colonies were observed at a frequency of 0.14%. Plasmids were purified from 10 positive clones and characterized by nucleotide sequence analysis. One of these, p~TUB1, contained an insert of approximately 1600 bp. Amino acid sequences predicted from the 5' and 3' nucleotide sequence of the p~TUB1 insert were highly homologous to the amino and carboxyl terminus sequences of known ~t-tubulins (see ref. 10), indicating that pctTUB1 probably encoded an intact H. contortus ~-tubulin. p~TUB1 was thus selected for further characterization. Southern hybridization analysis of H. contortus genomic DNA digests probed with the insert from pctTUB1 showed a complex banding pattern (Fig. 1). The number of hybridizing bands suggests that multiple introns and/or other closely related sequences are present. All eukaryotic organisms contain at least 2 ct-tubulin genes (see ref. 4), and it is likely that H. contortus contains one or more additional ~-tubulins. There was no hybridization to sheep genomic DNA digests under the
346
1
2
A
3
EI
B
Ad
E lAd
23.0 -
9.4 6.64.4-
28S 18SIll
I
2.3 2.01.8-
1 . 2 --
1.0Fig. 1. Hybridization analyses of H. contortus ~-tubulin. Left panel: Southern hybridization analysis. Genomic DNA from H. contortus was probed with the cDNA insert from p~TUB1 labeled with 32p by random priming as described [14]. Each lane contained 22 /~g gDNA digested with H i n d l l I (lane 1), PstI (lane 2) and SalI (lane 3). Molecular size markers (kb) are indicated on the left. Right panel: Northern hybridization analysis. Total RNA (7.5 #g) from egg stage (E), immature adults (21-day infections in sheep, I) and adult males (~>28-day infections, A~') was fractionated through 0.7% agarose gel and processed as described [14]. The filter was probed with (A) the 32p-labeled cDNA insert from FtTUB1 or (B) a cDNA encoding Ascaris suum actin [11].
conditions used (data not shown). This H. contortus a-tubulin is expressed throughout the developmental stages of H. contortus as shown by Northern hybridization analysis (Fig. !). A similar pattern of expression was observed with an Ascaris suum actin c D N A [11]. The plasmid paTUB1 was characterized by restriction enzyme analysis. Two E c o R I sites were found within the insert in addition to the flanking E c o R I sites in the pBluescript polylinker. Subclones of the c D N A insert were generated by digesting paTUB1 with E c o R I followed by treatment with T4 D N A ligase. The resulting mixture was used to transform E. coli strain DH5cc Clones containing plasmids representing all possible permutations of the
ligated E c o R I fragments were the source of D N A used in sequence analysis. The entire 1.6-kb insert was sequenced and was shown to contain an O R F of 1350 bp, which encodes a protein of 450 amino acids. The predicted amino acid sequence encoded by the ~TUB1 c D N A is very similar to other ~tubulins (Fig. 2), which are all closely related at the amino acid level [4,10]. The putative GTP binding site around position 148 ( G G G T G S G ) is completely conserved. Notable differences in the H. contortus gene compared to other ~tubulins include the presence of an arginine instead of the lysine found in all known ~tubulins at position 59 and the presence of a leucine and asparagine in the carboxyl terminal
347 Hc-Tuba Sm-Tuba2 Sm-Tubal Musatub H c - T u b a
MREVISIHIG
QAGVQIGNAC
...C..V.V
......
.. . C . . L . . . . . . . . • .
.C..V.V
......
TFFSETGSGR .......
A.K
Sm-Tubal
• .
.T.MT.
Musatub
• .
.C. . . A . K . .
NNYARGHYTI
GKEIIDLTLD
.............. .........
V..V
.......
Musatub
................
Hc-Tuba
SLLMERLSVD
V..V
N.V.
.............. SFMVDEAIY
N.H.ATNE
......
PV .....
.P.Q ................
A .................... DLTEFQTNLV
GLQGFLVFHS ........ ......
VVEPYNSILT
IGQIVSSITA
T ........ TFSPVISAEK
Sm-Tubal
.....................
YA.I
Musatub
.....................
YA ....................
Hc-Tuba
QMVKCDPRHG
KYMAVCLLFR
Musatub Hc-Tuba Sm-Tuba2 Sm-Tubal
.............. FKVGINYQPP
C.M.Y
..N.C...Y C...Y
GDVVPKDVNA .............. .............. VPRAVCMLSN
..................... ...........
Hc-Tuba
KRAFVHWYVG
EGMEEGEFSE
300
A .... A.
F ...........
S...TG. A .... A.
AIATIKTKRS
IQFVDWCPTG T
..........
N .... KT
..........
350
A ................ TTAIAEAWAR
LDHKFDLMYA
400
Q ....................
R .......
Q ............................ AREDLAALEK
Sm-Tuba2
.......................................
Sm-Tubal
............
Musatub
ITNMCFEPHN
Q ............................
A .........
.....................
......
....................
TWPGGDLAK
Musatub
250
S ..................
YA ....................
T.
200 T.
SLRFDGALNV
AYHEQLSVAE
.....................
........
THTTLEHSDC
T ........................... H.T ........ A ..................
Sm-Tuba2
..............
150
I .............
I .............................
PYPRIHFPLA
Sm-Tubal
FGGGTGSGFT
I .............
......................
RPSYTNLNRL
A .................... A ............ K..G.G
Hc-Tuba
K.S.Q
I00
Q ...............
...... IA ...................... I ..... IA.. I .................
S .....
Musatub
.... T
GLITGKEDAA
. . .KQ ...............
VYPAPQVSTA
DICRRNLDIE
Sm-Tuba2
TKEKI.GA.N TI..G
N
K...Q
50
.... N
.V.C
.V.KS..Q.S
YGKKAKLEFS
Hc-Tuba
SLGGCDDSFS TI..G
GTYRSLFHPE
RIRRLADNCT .....
V.E
.............. S .... A ......... E ...RS .... A
Musatub
Sm-Tuba2 Sm-Tubal
EPTVIDEIRT
..... FV ...........
Sm-Tubal
V
N .........
..... IFV ....
Sm-Tuba2
Sm-Tuba2 Sm-Tubal
M ......
...... FV ......
.K
QPDGQMPSDK
M ........................
HVPRAVMIDL
Sm-Tuba2
Hc-Tuba
WELYCLEHGI
M ........................
N ..... T .....
........................
T .......
DYEEVGVDSL
EDNGEEGDEY-449 V
A...C.TI
M ...........
I..Y
DGE..GEG.EY MLDEDDEEQEF . . E D . G E E - - -
Fig. 2. Comparison of the predicted amino acid sequence encoded by the peTUB1 c D N A with S. mansoni e-tubulin and mouse e4-tubulin. The H. contortus sequence (Hc-Tuba) is on the top line, the S. mansoni e-tubulin sequences Sin-Tuba2 [13] and Sm-Tubal [12] are in the middle and the mouse e-tubulin, Musatub [9], is on the bottom line. Identities between these sequences and the H. contortus e-tubulin are denoted by a dot, while differences are denoted by the corresponding single letter designation for the substituted amino acid.
region. Although the carboxy terminus is the most variable section of a-tubulins, these amino acids are rarely found there. The functional importance of these differences has not been defined. The H. contortus a-tubulin is quite similar to the mouse ~t4 isotype (92% amino acid identity over the first 440 residues). The S. m a n s o n i atubulin Sm-Tuba2, reported by Webster et al. [12], is also closely related, with 92% and 95% amino acid identity to the H . contortus and mouse e4 isotype, respectively, over the first 440 residues. In contrast, the S. mansoni atubulin Sm-Tubal, reported by Duvaux-Miret et al. [13], is much less closely related (82% amino acid identity to the H. contortus atubulin over the first 440 residues). Particular attention was paid to residues 33-48 in this S.
mansoni protein, which distinguish it from most metazoan ~-tubulins. This ~-tubulin is apparently not characteristic of other helminth ct-tubulins, which appear to diverge only slightly from typical metazoan ~-tubulins.
Acknowledgements We thank George Conder and Eileen Thomas for supplying H . contortus, Kathy Hiestand for preparation of the manuscript, Dr. Nick Cowan for the mouse ~t4-tubulin cDNA and Dr. Karen Bennett for the A s c a r i s suum actin cDNA.
348
References 1 Dustin, P. (1984) Microtubules. Springer-Verlag, New York. 2 Sullivan, K.F. (1988) Structure and utilization of tubulin isotypes. Annu. Rev. Cell Biol. 4, 687-716. 3 Lacey, E. (1988) The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. Int. J. Parasitol. 18, 885936. 4 Lacey, E. (1990) Mode of action of benzimidazoles. Parasitol. Today 6, 112 115. 5 Russell, G.J., Gill, J.H. and Lacey, E. (1992) Binding of [3H]benzimidazole carbamates to mammalian brain tubulin and the mechanism of selective toxicity of the benzimidazole anthelmintics. Biochem. Pharmacol. 43, 1095-1100. 6 Gill, J.H. and Lacey, E. (In Press) The kinetics of mebendazole binding to Haemonchus contortus tubulin. Int. J. Parasitol. 7 Geary, T.G., Nulf, S.C., Favreau, M.A., Tang, L., Prichard, R.K., Hatzenbuhler, N.T., Shea, M.H., Alexander, S.J. and Klein, R.D. (1992) Three/?-tubulin cDNAs from the parasitic nematode Haemonchus contortus. Mol. Biochem. Parasitol. 50, 295-305. 8 Klein, R.D., Olson, E.R., Favreau, M.A., Winterrowd, C.A., Hatzenbuhler, N.T., Shea, M.H., Nulf, S.C. and
Geary, T.G. (1991) Cloning of a cDNA encoding phosphofructokinase from H a e m o n c h u s contortus. Mol. Biochem. Parasitol. 48, 17-26. 9 Villasante, A., Wang, D., Dobner, P., Dolph, P., Lewis, S.A. and Cowan, N.J. (1986) Six mouse c~-tubulin mRNAs encode 5 distinct isotypes: testis-specific expression of two sister genes. Mol. Cell. Biol. 6, 2409-2419. 10 Little, M. and Seehaus, T. (1988) Comparative analysis of tubulin sequences. Comp. Biochem. Physiol. 90B, 655. 11 Bennett, K.L. and Ward, S. (1986) Neither a gene linespecific nor several somatically expressed genes are lost or rearranged during embryonic chromatin dismutation in the nematode Ascaris lumbricoides var. suum. Dev. Biol. 118, 141-147. 12 Webster, P.J., Seta, K.A., Chung, S.C. and Mansour, T.E. (1992) A cDNA encoding an c~-tubulin from Schistosoma mansoni. Mol. Biochem. Parasitol. 51, 169 170. 13 Duvaux-Miret, O., Baratte, B., Dissous, C. and Capron, A. (1991) Molecular cloning and sequencing of the ~-tubulin gene from Schistosoma mansoni. Mol. Biochem. Parasitol. 49, 337 340. 14 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
345
MOLBIO 01868
Short C o m m u n i c a t i o n
Cloning of a cDNA encoding
-tubulin from Haemonchus contortus
R o n a l d D. Klein a, Susan C. N u l f b, Susan J. A l e x a n d e r - B o w m a n b, C a r o l y n B. M a i n o n e a, Christal A. W i n t e r r o w d b and T i m o t h y G. G e a r y aMolecular Biology Research and bAnimal Health Therapeutics Research, Upjohn Laboratories, The Upjohn Company, Kalamazoo, MI, USA (Received 12 June 1992; accepted 8 September 1992)
Key words: ct-tubulin cDNA; Haemonchus contortus; Nematode
Microtubules, which are polymers of dimers of ct- and fl-tubulin, have diverse roles in cellular morphology, axonal transport and cell division [1]. Most eukaryotic organisms express multiple, distinct isotypes of ~- and fltubulin, though specific functional differences among these isotypes have generally not been discerned (see ref. 2 for review). A particularly intriguing aspect of tubulin in nematodes is that the benzimidazole class of anthelmintics acts to destabilize microtubules by disrupting the ~-fl dimer (see refs. 3 and 4). This interaction is selective for nematode tubulin [5,6], which explains the chemotherapeutic utility of these drugs. The molecular basis for this selective toxicity is not yet clear. In order to better understand the molecular pharmacology of these important drugs, we have cloned and characterized 3 fl-tubulin cDNAs from H a e m o n c h u s contortus, a parasite of the ruminant abomasum [7]. In this report, we describe an ~-tubulin cDNA cloned from the Correspondence address: R.D. Klein, 7242/267/5, The Upjohn Company, Kalamazoo, MI 49007, USA. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankT M data base with the accession number L-02108. Abbreviations: SSC, standard saline citrate; ORF, open reading frame.
same organism. An immature adult H. contortus cDNA library in the vector 2ZAPII [8] was plated at a density of 5 x 104 pfu per plate and screened with a mouse ~4 tubulin cDNA (ref. 9; a gift from N. Cowan, New York University) at 65°C in 0.5 x SSC. Hybridizing colonies were observed at a frequency of 0.14%. Plasmids were purified from 10 positive clones and characterized by nucleotide sequence analysis. One of these, p~TUB1, contained an insert of approximately 1600 bp. Amino acid sequences predicted from the 5' and 3' nucleotide sequence of the p~TUB1 insert were highly homologous to the amino and carboxyl terminus sequences of known ~t-tubulins (see ref. 10), indicating that pctTUB1 probably encoded an intact H. contortus ~-tubulin. p~TUB1 was thus selected for further characterization. Southern hybridization analysis of H. contortus genomic DNA digests probed with the insert from pctTUB1 showed a complex banding pattern (Fig. 1). The number of hybridizing bands suggests that multiple introns and/or other closely related sequences are present. All eukaryotic organisms contain at least 2 ct-tubulin genes (see ref. 4), and it is likely that H. contortus contains one or more additional ~-tubulins. There was no hybridization to sheep genomic DNA digests under the
346
1
2
A
3
EI
B
Ad
E lAd
23.0 -
9.4 6.64.4-
28S 18SIll
I
2.3 2.01.8-
1 . 2 --
1.0Fig. 1. Hybridization analyses of H. contortus ~-tubulin. Left panel: Southern hybridization analysis. Genomic DNA from H. contortus was probed with the cDNA insert from p~TUB1 labeled with 32p by random priming as described [14]. Each lane contained 22 /~g gDNA digested with H i n d l l I (lane 1), PstI (lane 2) and SalI (lane 3). Molecular size markers (kb) are indicated on the left. Right panel: Northern hybridization analysis. Total RNA (7.5 #g) from egg stage (E), immature adults (21-day infections in sheep, I) and adult males (~>28-day infections, A~') was fractionated through 0.7% agarose gel and processed as described [14]. The filter was probed with (A) the 32p-labeled cDNA insert from FtTUB1 or (B) a cDNA encoding Ascaris suum actin [11].
conditions used (data not shown). This H. contortus a-tubulin is expressed throughout the developmental stages of H. contortus as shown by Northern hybridization analysis (Fig. !). A similar pattern of expression was observed with an Ascaris suum actin c D N A [11]. The plasmid paTUB1 was characterized by restriction enzyme analysis. Two E c o R I sites were found within the insert in addition to the flanking E c o R I sites in the pBluescript polylinker. Subclones of the c D N A insert were generated by digesting paTUB1 with E c o R I followed by treatment with T4 D N A ligase. The resulting mixture was used to transform E. coli strain DH5cc Clones containing plasmids representing all possible permutations of the
ligated E c o R I fragments were the source of D N A used in sequence analysis. The entire 1.6-kb insert was sequenced and was shown to contain an O R F of 1350 bp, which encodes a protein of 450 amino acids. The predicted amino acid sequence encoded by the ~TUB1 c D N A is very similar to other ~tubulins (Fig. 2), which are all closely related at the amino acid level [4,10]. The putative GTP binding site around position 148 ( G G G T G S G ) is completely conserved. Notable differences in the H. contortus gene compared to other ~tubulins include the presence of an arginine instead of the lysine found in all known ~tubulins at position 59 and the presence of a leucine and asparagine in the carboxyl terminal
347 Hc-Tuba Sm-Tuba2 Sm-Tubal Musatub H c - T u b a
MREVISIHIG
QAGVQIGNAC
...C..V.V
......
.. . C . . L . . . . . . . . • .
.C..V.V
......
TFFSETGSGR .......
A.K
Sm-Tubal
• .
.T.MT.
Musatub
• .
.C. . . A . K . .
NNYARGHYTI
GKEIIDLTLD
.............. .........
V..V
.......
Musatub
................
Hc-Tuba
SLLMERLSVD
V..V
N.V.
.............. SFMVDEAIY
N.H.ATNE
......
PV .....
.P.Q ................
A .................... DLTEFQTNLV
GLQGFLVFHS ........ ......
VVEPYNSILT
IGQIVSSITA
T ........ TFSPVISAEK
Sm-Tubal
.....................
YA.I
Musatub
.....................
YA ....................
Hc-Tuba
QMVKCDPRHG
KYMAVCLLFR
Musatub Hc-Tuba Sm-Tuba2 Sm-Tubal
.............. FKVGINYQPP
C.M.Y
..N.C...Y C...Y
GDVVPKDVNA .............. .............. VPRAVCMLSN
..................... ...........
Hc-Tuba
KRAFVHWYVG
EGMEEGEFSE
300
A .... A.
F ...........
S...TG. A .... A.
AIATIKTKRS
IQFVDWCPTG T
..........
N .... KT
..........
350
A ................ TTAIAEAWAR
LDHKFDLMYA
400
Q ....................
R .......
Q ............................ AREDLAALEK
Sm-Tuba2
.......................................
Sm-Tubal
............
Musatub
ITNMCFEPHN
Q ............................
A .........
.....................
......
....................
TWPGGDLAK
Musatub
250
S ..................
YA ....................
T.
200 T.
SLRFDGALNV
AYHEQLSVAE
.....................
........
THTTLEHSDC
T ........................... H.T ........ A ..................
Sm-Tuba2
..............
150
I .............
I .............................
PYPRIHFPLA
Sm-Tubal
FGGGTGSGFT
I .............
......................
RPSYTNLNRL
A .................... A ............ K..G.G
Hc-Tuba
K.S.Q
I00
Q ...............
...... IA ...................... I ..... IA.. I .................
S .....
Musatub
.... T
GLITGKEDAA
. . .KQ ...............
VYPAPQVSTA
DICRRNLDIE
Sm-Tuba2
TKEKI.GA.N TI..G
N
K...Q
50
.... N
.V.C
.V.KS..Q.S
YGKKAKLEFS
Hc-Tuba
SLGGCDDSFS TI..G
GTYRSLFHPE
RIRRLADNCT .....
V.E
.............. S .... A ......... E ...RS .... A
Musatub
Sm-Tuba2 Sm-Tubal
EPTVIDEIRT
..... FV ...........
Sm-Tubal
V
N .........
..... IFV ....
Sm-Tuba2
Sm-Tuba2 Sm-Tubal
M ......
...... FV ......
.K
QPDGQMPSDK
M ........................
HVPRAVMIDL
Sm-Tuba2
Hc-Tuba
WELYCLEHGI
M ........................
N ..... T .....
........................
T .......
DYEEVGVDSL
EDNGEEGDEY-449 V
A...C.TI
M ...........
I..Y
DGE..GEG.EY MLDEDDEEQEF . . E D . G E E - - -
Fig. 2. Comparison of the predicted amino acid sequence encoded by the peTUB1 c D N A with S. mansoni e-tubulin and mouse e4-tubulin. The H. contortus sequence (Hc-Tuba) is on the top line, the S. mansoni e-tubulin sequences Sin-Tuba2 [13] and Sm-Tubal [12] are in the middle and the mouse e-tubulin, Musatub [9], is on the bottom line. Identities between these sequences and the H. contortus e-tubulin are denoted by a dot, while differences are denoted by the corresponding single letter designation for the substituted amino acid.
region. Although the carboxy terminus is the most variable section of a-tubulins, these amino acids are rarely found there. The functional importance of these differences has not been defined. The H. contortus a-tubulin is quite similar to the mouse ~t4 isotype (92% amino acid identity over the first 440 residues). The S. m a n s o n i atubulin Sm-Tuba2, reported by Webster et al. [12], is also closely related, with 92% and 95% amino acid identity to the H . contortus and mouse e4 isotype, respectively, over the first 440 residues. In contrast, the S. mansoni atubulin Sm-Tubal, reported by Duvaux-Miret et al. [13], is much less closely related (82% amino acid identity to the H. contortus atubulin over the first 440 residues). Particular attention was paid to residues 33-48 in this S.
mansoni protein, which distinguish it from most metazoan ~-tubulins. This ~-tubulin is apparently not characteristic of other helminth ct-tubulins, which appear to diverge only slightly from typical metazoan ~-tubulins.
Acknowledgements We thank George Conder and Eileen Thomas for supplying H . contortus, Kathy Hiestand for preparation of the manuscript, Dr. Nick Cowan for the mouse ~t4-tubulin cDNA and Dr. Karen Bennett for the A s c a r i s suum actin cDNA.
348
References 1 Dustin, P. (1984) Microtubules. Springer-Verlag, New York. 2 Sullivan, K.F. (1988) Structure and utilization of tubulin isotypes. Annu. Rev. Cell Biol. 4, 687-716. 3 Lacey, E. (1988) The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. Int. J. Parasitol. 18, 885936. 4 Lacey, E. (1990) Mode of action of benzimidazoles. Parasitol. Today 6, 112 115. 5 Russell, G.J., Gill, J.H. and Lacey, E. (1992) Binding of [3H]benzimidazole carbamates to mammalian brain tubulin and the mechanism of selective toxicity of the benzimidazole anthelmintics. Biochem. Pharmacol. 43, 1095-1100. 6 Gill, J.H. and Lacey, E. (In Press) The kinetics of mebendazole binding to Haemonchus contortus tubulin. Int. J. Parasitol. 7 Geary, T.G., Nulf, S.C., Favreau, M.A., Tang, L., Prichard, R.K., Hatzenbuhler, N.T., Shea, M.H., Alexander, S.J. and Klein, R.D. (1992) Three/?-tubulin cDNAs from the parasitic nematode Haemonchus contortus. Mol. Biochem. Parasitol. 50, 295-305. 8 Klein, R.D., Olson, E.R., Favreau, M.A., Winterrowd, C.A., Hatzenbuhler, N.T., Shea, M.H., Nulf, S.C. and
Geary, T.G. (1991) Cloning of a cDNA encoding phosphofructokinase from H a e m o n c h u s contortus. Mol. Biochem. Parasitol. 48, 17-26. 9 Villasante, A., Wang, D., Dobner, P., Dolph, P., Lewis, S.A. and Cowan, N.J. (1986) Six mouse c~-tubulin mRNAs encode 5 distinct isotypes: testis-specific expression of two sister genes. Mol. Cell. Biol. 6, 2409-2419. 10 Little, M. and Seehaus, T. (1988) Comparative analysis of tubulin sequences. Comp. Biochem. Physiol. 90B, 655. 11 Bennett, K.L. and Ward, S. (1986) Neither a gene linespecific nor several somatically expressed genes are lost or rearranged during embryonic chromatin dismutation in the nematode Ascaris lumbricoides var. suum. Dev. Biol. 118, 141-147. 12 Webster, P.J., Seta, K.A., Chung, S.C. and Mansour, T.E. (1992) A cDNA encoding an c~-tubulin from Schistosoma mansoni. Mol. Biochem. Parasitol. 51, 169 170. 13 Duvaux-Miret, O., Baratte, B., Dissous, C. and Capron, A. (1991) Molecular cloning and sequencing of the ~-tubulin gene from Schistosoma mansoni. Mol. Biochem. Parasitol. 49, 337 340. 14 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
