Báo cáo Y học: Dystrobrevin requires a dystrophin-binding domain to function in Caenorhabditis elegans

Eur. J. Biochem. 269, 1607–1612 (2002) Ó FEBS 2002 Dystrobrevin requires a dystrophin-binding domain to function in Caenorhabditis elegans Karine Grisoni, Kathrin Gieseler and Laurent Segalat CGMC, CNRS-UMR, Universite Lyon, Villeurbanne, France Dystrobrevin is one of the intracellular components of the transmembrane dystrophin–glycoprotein complex (DGC). The functional role of this complex in normal and patho-logical situations has not yet been clearly established. Dystrobrevin disappears from the muscle membrane in Duchenne muscular dystrophy (DMD), which results from dystrophin mutations, as well as in limb girdle muscular dystrophies (LGMD), which results from mutations affect-ing other members of the DGC complex. These findings thereforesuggestthatdystrobrevinmayplayapivotalrolein the progression of these clinically related diseases. In this study, we used the Caenorhabditis elegans model to address the question of the relationship between dystrobrevin binding to dystrophin and dystrobrevin function. Deletions Duchenne muscular dystrophy (DMD) is an inherited muscular disease in which the patients’ muscles gradually degenerate. So far, no treatment exists for DMD. The diseaseiscausedbymutationsaffectingthedystrophingene, which encodes a 3685-amino-acid protein (reviewed in [1]). Dystrophin is a submembrane protein associated with a transmembrane dystrophin–glycoprotein complex (DGC) comprising dystroglycans, sarcoglycans, sarcospan, syntro-phins and dystrobrevins [1–3]. DGC proteins have attracted an increasing amount of attention over the last few years, because they might help to explain the physiopathology of the disease, and may also provide therapeutic clues. Dystrobrevins form a family of proteins that are unique in that they are both dystrophin-associated proteins, and homologous to the C-terminal region of dystrophin. Alpha-dystrobrevin was originally identified as a molecule that copurifies with nicotinic acetylcholine receptors in sucrose gradients[4,5].Itwaslaterrecognizedasoneoftheproteins, which associates with dystrophin to form the dystrophin– glycoprotein complex (DGC) [4,6,7]. A second dystro-brevin, b-dystrobrevin, is mainly expressed in nerve tissues Correspondence toL. Segalat,CGMC, UniversiteLyon1, 43 bld du 11 Novembre, 69622 Villeurbanne cedex, France. Fax: + 33 4 72 44 05 55, Tel.: + 33 4 72 43 29 51, E-mail: segalat@maccgmc.univ-lyon1.fr Abbreviations: DGC, dystrophin–glycoprotein complex; DMD, Duchenne muscular dystrophy; LGMD, limb girdle muscular dystrophy; nNOS, neuronal nitric oxide synthase; AD, activation domain; DNA-BD, DNA binding domain; SD, synthetic dropout medium; SBR, syntrophin binding region. (Received 16 October 2001, revised 10 January 2002, accepted 11 January 2002) of the dystrobrevin protein were performed and the ability of the mutated forms to bind to dystrophin was tested both in vitro and in a two-hybrid assay, as well as their ability to rescue dystrobrevin (dyb-1) mutations in C. elegans. The deletions affecting the second helix of the Dyb-1 coiled-coil domainabolishedthebinding ofdystrobrevintodystrophin both in vitro and in the two-hybrid assay. These deletions alsoabolishedtherescuingactivityofafunctionaltransgene invivo.Theseresultsareconsistentwithamodelaccordingto which dystrobrevin must bind to dystrophin to be able to function properly. Keywords: dystrophin; dystrobrevin; nematode; Caeno-rhabditis elegans. [8,9]. Mice carrying a knockout mutation of the a-dystrobrevin gene (adbn mice) suffer from a cardiac and skeletal muscle myopathy reminiscent of dystrophin (mdx) mutations [10]. a-Dystrobrevin binds to dystrophin via a coiled-coiled motif present in both proteins, and to the PDZ domain containing syntrophins [11,12]. Indirect evidence suggests that dystrobrevin may also bind to other members of the DGC [13]. Although no enzymatic activity has yet been assigned to dystrobrevins, there are several indications that they may play a role in signalling mechanisms. First, dystrobrevins are tyrosine-phosphorylated proteins [5,14]. Secondly, in the absence of a-dystrobrevin, the signalling molecule, neuronalnitric oxidesynthase(nNOS)disappears from the muscular membrane [10]. In addition, two lines of evidence suggest that dystro-brevin may play a key role in the muscle degeneration observed in DMD and sarcoglycanopathies; first, dystro-brevin immunostaining decreases greatly in DMD and in several sarcoglycanopathies [15]. Secondly, although the DGC components (with the exception of NOS) are not affectedbytheabsenceofdystrobrevininadbnmice,muscle degeneration occurs. The nematode Caenorhabditis elegans has homologues of most of the DGC proteins (L. Segalat, unpublished results). There is one dystrophin- and one dystrobrevin-like gene in the genome of C. elegans (dys-1 and dyb-1, respectively) [16,17]. C. elegans dystrophin and dystrobrevin are able to bind to each other in vitro [18] in the same way as their mammalian counterparts [12], and they also bind to syntrophin [18]. dys-1 and dyb-1 mutants display a similar behavioural phenotype consisting of hyperactivity, exagger-ated bending of the head when moving forward, and a tendency to hypercontract [16,17]. In addition, progressive muscle degeneration is observed when dys-1 or dyb-1 1608 K. Grisoni et al. (Eur. J. Biochem. 269) mutations are introduced in a sensitized hlh-1(cc561) genetic background that makes C. elegans muscles fragile [19,20]. In this study, we addressed the question as to whether the ability of dystrobrevin to function properly may depend on its association with dystrophin. First, we refined the dystrophin-binding region on dystrobrevin (Dyb-1) by performing deletion-mapping experiments in vitro. We then tested the ability of the truncated Dyb-1 proteins to bind to dystrophin (Dys-1) in a yeast two-hybrid assay, as well as their ability to rescue dyb-1 mutants. EXPERIMENTAL PROCEDURES Construction of deleted forms of Dyb-1 for in vitro binding experiments Deletions were carried out on the dyb-1 coding sequence, using clone AN450 [encoding Dyb-1 amino acids 390–543 fused in frame to the GST coding sequence; plasmid pGEX 3X (Pharmacia)] [18]. AN450 DNA (500 ng) was cut with the restriction enzyme MfeI. The cut DNA was then distributed among several tubes incubated with 0.05 lL of BAL31 exonuclease for various times (typically 0–10 min). The reactions were stopped by adding EGTA to 4 mM and heatingat65 °Cfor10 min.DNAwaspurifiedonaWizard column(Promega)andtheactionofBAL31wascheckedby loading an aliquot of each tube onto an agarose gel column. The DNA corresponding to the deletions required was treated by applying T4 DNA polymerase in the presence of nucleotides to create blunt ends, which were ligated and the plasmids were transformed in Escherichia coli DH5. Clones were picked randomly and analysed using sequencing procedures. Any clones carrying a frame shift were rejected. Construct 6¢4 was built using similar procedures, but using the enzyme HindIII instead of MfeI. The amino acids removed in the deletions were 489–499 (clone 2¢5), 487–513 (clone5¢1),489–528(clone5¢2B),471–517(clone5¢5B),478– 543 (clone 2¢1), and 391–450 (clone 6¢4). Clones 2¢1 and 6¢4 have been described previously [18], but clone 2¢1 was erroneously reported to be deleted in amino acids 478–521. This correction makes no difference to the interpretation of our previous data. In vitro interactions Constructs were transformed into the E. coli strain BL21 DE3 and the fusion proteins were produced as follows. After cell sonication and centrifugation, the supernatant was loaded onto glutathione–Sepharose beads (Pharma-cia). Approximately 20 lg of resin bound proteins were washed in binding buffer [Hepes 20 mM pH 7.4, KOAc 110 mM, NaOAc 5 mM, Mg(OAc) 2.5 mM, NP40 0.05%, dithiothreitol 1 mM, leupeptin 10 mgÆmL)1, aprotinin 10 mgÆmL)1, pepstatin 10 mgÆmL)1, phenylmethanesulfo-nyl fluoride (1 mM)]. The 35S-labelled Dys-1 C-terminal end was synthesized using a coupled in vitro transcription and translation kit (Promega) with cDNA yk12c11 [16]. The preparations were incubated for 2 h at 4 °C. GST controls were performed using 1–2 times the amount of fusion protein. After five washes with binding buffer, the labelled proteins were eluted by boiling the preparation for 3 min in gel loading buffer. Gels were dried, exposed Ó FEBS 2002 overnight and revealed using a radiographic analyser (Fuji BAS-1500). Band intensity was quantitated using the analyser software on at least three independent experiments. Constructs for the yeast two-hybrid assay The C-terminal end of Dys-1 (amino acids 2857–3674) was fused to the DNA binding domain (DNA-BD) of the Gal4 protein. For this purpose, a 2,4 kb dys-1 cDNA fragment (yk12c11) was cloned into the polylinker of pAS2-1 (Clontech) with respect to the reading frame. Dyb-1fragmentsanddeletionswerePCRamplifiedusing clones AN 450, 2¢5, 5¢1, 5¢2B, 5¢5B and 6¢4 in pGEX 3X as templates (see below) and cloned into pACT2 (Clontech) in frame with the activation domain (AD) of the Gal4 protein and the HA epitope. The resulting constructs were called AD-AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, and AD-6¢4, respectively. All DNA constructs were checked by performing DNA sequencing. Yeast two-hybrid analysis Construct DNA-BD-Dys-1 was transformed into the yeast strain CG 1945 using the LiAc transformation procedure (Clontech, Yeast protocols Handbook, PT 3024-1). Trans-formants were selected on synthetic dropout (SD) media (Clontech) minus tryptophan. A DNA-BD-Dys-1 expressing yeast strain was selected and transformed with plasmids AD-AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, or AD-6¢4. Transformants were selected on SD media minus tryptophan and leucin. Interactions between the DNA-BD-Dys-1 protein and the various forms of the AD-Dyb-1 fusion proteins were analysed on the basis of transactivation of the HIS3 reporter gene after 3 days of growth on SD medium devoid of tryptophan, leucin and histidin. A strain carrying both theDNA-BD-Dys-1proteinandtheemptypACT2plasmid was used as a negative control. Western blots with yeast protein extracts For Western blot analysis, yeast protein extracts were prepared from strains carrying both DNA-BD-Dys-1 plasmids and AD-Dyb-1 plasmids (AN450, AD-2¢5, AD-5¢1, AD-5¢2B, AD-5¢5B, or AD-6¢4). Overnight cul-tures (5-mL) were prepared in SD media minus trypto-phan and leucin. The next day, 1 mL of overnight culture was transferred into 10 mL of YPD medium. The diluted culture was incubated for several hours at 30 °C until D600 ˆ 0.3 for 1 mL. Cells (3 D600 units) were spun down and frozen at )70 °C for at least one hour. The yeast pellet was resuspended in 60 lL of sample buffer [21]. After boiling the mixture for 5 min, and centrifuging for 30 s at 13 000 g, 10 lL of supernatant was loaded onto each lane of a 0.1% SDS/10% polyacrylamide gel. Proteins were transferred onto a BA83 nitrocellulose membrane (Schleicher & Schuell) in transfer buffer (Tris 25 mM, glycine 190 mM, SDS 0.01%, ethanol 20%) for 1 h at 100 V. AD-Dyb-1 fusion proteins were detected using a rabbit polyclonal anti-(Dyb-1) Ig [19] at a dilution 1 : 500. Peroxidase-coupled anti-(rabbit IgG) Ig (Biorad) was used at a dilution of 1 : 3000. Blots Ó FEBS 2002 Dystrophin–dystrobrevin interactions in C. elegans (Eur. J. Biochem. 269) 1609 Fig. 1. Deletionsusedinthisstudy.The‘WT’linerepresentstheamino-acid sequence of the wild-type Dyb-1 protein in the predicted coiled-coil domain region. The predicted helices forming the domain are shown by hatched boxes. Numbers above the wild-type sequence indicatetheamino-acidcoordinatesofthehelices.Deletionsareshown below the wild-type sequence. Numbers indicate the coordinates of the breakpoints. Deletions were generated by exonuclease digestion. Note that the 6¢4 deletion extends on the left side further than shown on the drawing. For in vitro binding experiments, the corresponding DNAs were cloned into the pGEX vector to produce Dyb-1–GST fusion proteins [18]. The right column gives the binding anity of the various constructs to 35S-labelled Dys-1 in arbitrary units (mean ‹ SD). One unit is defined as the autoradiogram intensity obtained with the neg-ativecontrolGST.Asterisksindicatevaluessignificantlydifferentfrom wild-type. Constructs 5¢1, 5¢5B and 2¢1 have significantly reduced anity to Dys-1. Fig. 2. In vitro binding of Dyb-1 (dystrobrevin) to Dys-1 (dystrophin). Representativeexampleofinvitrobindingexperiments.Thesamegelis shown in Coomassie staining (top) and autoradiography (bottom). The gel was loaded with various GST–Dyb-1 fusion proteins (and GST alone) after incubation with equal amounts of in vitro translated 35S-labelled DYS-1. The signal intensity of the autoradiogram was quantitated with a radiographic analyser (Biorad). MW, Molecular mass markers. T, aliquot of the in vitro translation product. were revealed using the ECL+ kit (Amersham) as recommended by the supplier. RESULTS Functional assay in C. elegans First, a dyb-1 functional construct was obtained by modi-fying a previously built dyb-1:gfp construct [19]. The dyb-1:gfp construct, which has been previously described, was shortened on the 5¢ end to leave 2.9 kb of upstream sequence,andvariousrestrictionenzymesiteswereremoved and added by performing synonymous point mutations to yield the constructdyb-1:gfp VII, which has single AgeI and MluI sites at codons 390 and 543. This construct encodes a functionalDyb-1geneasitcanrescuedyb-1mutations(data not shown). Secondly, Dyb-1 construct AN450 and the deletion derivatives described above were transferred from pGEX into dyb-1:gfp VII using PCR-amplifying procedures with primers carrying AgeI and MluI sites, and cloned into the single AgeI and MluI sites of dyb-1:gfp VII (Fig. 5). Positives clones were checked by determining their seq-uence. dyb-1:gfp VII and constructs carrying either AN450 or the deletions were injected at a concentration of 1 ngÆlL)1 along with the transformation marker KP13 [22] using standard procedures [23] into worms carrying the putativenullalleledyb-1(cx36)[17].Transgenicstrainswere grown at 23 °C. Mapping of the dystrophin-binding site on Dyb-1 The results of a previous study suggested that the Dys-1-binding region on Dyb-1 was located in the second helix of the predicted coiled-coil domain [18]. We refined this analysis by creating additional deletions by random muta-genesis and testing their affinity for Dys-1. Clones 2¢5, 5¢1, 5¢2B, and 5¢5B were obtained by inducing exonuclease digestion of the reference clone AN450, which encodes the amino acids 390–543 of Dyb-1 fused to the GST protein [18]. These four clones contain various breakpoints within the second helix of the predicted coiled-coil domain (H2) (Fig. 1). The deleted amino acids were 489–499 (clone 2¢5), 487–513 (clone 5¢1), 489–528 (clone 5¢2B) and 471–517 (clone 5¢5B). Clone 2¢1, lacking amino acids 478–543, was used as a negative control [18]. Clone 6¢4, lacking amino acids 391–450, was used as a second positive control [18]. The constructs were used to produce Dyb-1–GST chimeric proteins in E. coli, which were affinity purified on gluthati-one–Sepharose beads and subjected to in vitro binding with 35S-labelled Dys-1. Clones 2¢5 and 5¢2B bound to Dys-1 at levels that were not significantly different from those of the positivecontrols AN450 (Fig. 2)and 6¢4 (gelnotshown).In contrast, the binding activity of clones 5¢1 and 5¢5B was weaker (Fig. 2). The difference between clones 2¢5, 5¢1 and 1610 K. Grisoni et al. (Eur. J. Biochem. 269) Fig. 3. WesternblotofDyb-1deletionsproducedinyeast.Westernblots were prepared with protein extracts of yeast carrying DNA-BD-Dys-1 plasmids and empty pACT2 (lane 1), AD-AN450 (lane 2), AD-2¢5 (lane 3), AD-5¢1 (lane 4), AD-5¢2B (lane 5), AD-5¢5B (lane 6) and AD-6¢4 (lane 7). The blot was probed with mouse monoclonal antibodies directed against the HA epitope situated between the C-terminal end of the activation domain of the Gal4 protein and the various Dyb-1 proteins. Molecular mass standards are shown on the right. 5¢2B is of interest because these clones have left breakpoints differing by only two amino acids. Although clones 2¢5 and 5¢2B (cutting at position 489) display a wild-type pattern of binding behaviour, clone 5¢1 (cutting at position 487) does not.Therefore,thethirdheptadrepeatofthesecondhelixof Dyb-1 (amino acids 484–490) seems to be critical for proper Dys-1 binding to occur in vitro. Dys-1/Dyb)1 interactions in the yeast two hybrid assay Next, we tested the ability of the various forms of Dyb-1 to interact with Dys-1 in a two-hybrid assay. The Dyb-1 control and mutant clones were fused to the activation domain of the Gal4 yeast transcription factor and were tested against the entire C-terminal end of Dys-1 (amino acids 2857–3674) fused to the DNA-binding domain of Gal4. The expression of wild-type and truncated proteins was checked using the Western blotting procedure. This confirmed that all the fusion proteins were correctly expressed and that the experiment was not biased by any differences in the protein expression levels (Fig. 3). Among the six constructs tested, only the wild-type Dyb-1 fragment andthe6¢4fragmentresultedinthegrowthofyeastsonHis-plates, which can occur only if Dyb-1 binds to Dys-1 (Fig. 4). Similar results were obtained when a shorter Dys-1 fragment (amino acids 3402–3674) encompassing the syn-trophin-binding domain and the coiled-coil domain (cor-responding to BB810 in [18]) was used (not shown). These results indicate that all the deletions affecting the second helix of Dyb-1, including the shortest deletion (clone 2¢5), greatly reduce the interactions between Dys-1 and Dyb-1 in the yeast system. Functional complementation of Dyb-1 deletions in C. elegans The only functional assay available for dystrobrevin resides in functional complementation. To test whether the dele-tionsofvariouspartsofthecoiled-coildomainhadaneffect on the in vivo function of Dyb-1, we created transgenes Ó FEBS 2002 Fig. 4. Yeast two-hybrid assay. A plate containing SD media minus leucin, tryptophan and histidine was seeded with yeast carrying DNA-BD-Dys-1 and various AD-Dyb-1 plasmids or empty pACT2 (as a negativecontrol),andincubatedat30 °Cfor3days.Growthonmedia devoid of histidine can occur only if Dys-1 and Dyb-1 interact and the HIS3reportergeneistransactivated.Onlythewild-typeconstructAD-AN450 and the AD-6¢4 construct promoted growth in this assay. The other constructs, all carrying deletions in the second helix of the Dyb-1 coiled-coil domain (H2), were unable to promote growth in this assay, which indicates that the H2 helix is a critical prerequisite for Dys-1/ Dyb-1 interactions to be possible. carryingthesamedeletionsasthosetestedinvitroandinthe yeast system. We transferred the deletions into the vector dyb-1:gfp VII, which is a functional transgene consisting of genomic Dyb-1 sequences (Fig. 5). Because the deletions are derivatives of clone AN450, a cDNA fragment that encompasses several exons, it was necessary first to check whether removing introns 7 and 8 had any effect on the rescuing activity of dyb-1:gfp VII. When the 1.2-kb AgeI– MluI genomic fragment of dyb-1:gfp VII was replaced by the 450-bp AN450 cDNA fragment encoding the same amino acids (Fig. 5), rescue of dyb-1(cx36) animals still occurred in two out of three lines transgenic lines (Table 1), whichindicatesthatremovingintrons7and8didnotimpair the rescuing capacity of dyb-1:gfp VII. Then we tested the various deletions. Three out of the four lines obtained with deletion 6¢4showedconsistent,althoughonly partial,rescue (Table 1). In these lines, the dyb-1 behavioural phenotype (head bending and hyperlocomotion) was intermediate betweenmutantandwild-type.Thisindicatesthat,although deleting the syntrophin binding region (SBR) and the first helix reduces the activity of the protein, it remains partly functional. Five lines were obtained with deletion 2¢5 (the shortest deletion affecting the second helix); only one out of thefivelinestestedshowedaweakrescuingeffect,whichwas far less conspicuous than that observed with construct 6¢4 (Table 1). Worms carrying the remaining constructs (5¢1, 5¢2B and 5¢5B) did not display any visible rescue. All in all, these data indicate that deletions affecting the second helix oftheDyb-1coiled-coildomainstronglydecreaseorabolish the functional properties of Dyb-1. DISCUSSION The aim of this study was twofold: first, to confirm and refine the localization of Dys-1 binding sites on Dyb-1, and secondly to test whether there may exist a correlation Ó FEBS 2002 Dystrophin–dystrobrevin interactions in C. elegans (Eur. J. Biochem. 269) 1611 Fig. 5. Drawing of the constructs used for in vivo complementation tests. Top, map of the dyb-1:gfp VII construct. dyb-1:gfp VII is a 8-kb genomic fragment of the dyb-1 gene cloned into a pGEM backbone, in which the gfp coding sequence has been added termin-ally to the dyb-1 coding sequence. Bottom: map of the constructs used for the in vivo experiments. The various deletions are deri-vatives of the AN450 construct, a fragment of dyb-1 cDNA cloned into the GST-containing vector pGEX. Deleted regions are indicated in dark. The deletions were transfered into dyb-1:gfp VII by PCR using the unique AgeI and MluI restriction sites of dyb-1:gfp VII. As a result, introns 7 and 8 of dyb-1:gfp VII were removed in these constructs. These construct were injected in dyb-1(cx36) mutants to assay their ability to rescue the mutant phenotype. Table 1. Rescuing activity of Dyb-1 deletions. Constructs were injected in dyb-1(cx36) animals along with the transformation marker KP13 [22]. dyb-1(cx36) animals display a behavioral phenotype consisting of hyperactivity, exaggerated bending of the head when moving forward, and a tendency to hypercontract when moving backwards. + + +, transgenic animals not distinguishable from wild-type animals; + +, transgenic animalsresemblewild-typebutremainslightlyhyperactiveandbendtheirheadmorethanwild-type; +,transgenicanimalsremainhyperactiveand bend their head,but can be distinguishedfrom nontransgenic siblings in blind tests; ‹, some transgenic animals showa slightly improvedbehavior but transgenics cannot be recognized with certainty in blind tests; –, no modification of the phenotype could be observed. Construct dyb-1:gfp VII AN450 in dyb-1:gfp VII 2¢5 in dyb-1:gfp VII 5¢1 in dyb-1:gfp VII 5¢2B in dyb-1:gfp VII 5¢5B in dyb-1:gfp VII 6¢4 in dyb-1:gfp VII Number of transgenic lines 3 3 5 6 4 8 4 Number of rescuing lines 2 2 1 0 0 0 3 Rescue in best line(s) + + + + + ‹ – – – + between the binding of dystrobrevin to dystrophin and functional activity of dystrobrevin. The C. elegans model organism was particularly well suited for the latter part because transgenic animals can be quickly obtained in this species. As long as the catalytic, enzymatic, or other functional activity of the dystrobrevin protein will remain unknown, the only way of making functional investi-gations will continue to be through complementation of mutations. A preliminary in vitro study on Dys-1–Dyb-1 interactions pointed to the second helix (H2) of the predicted coiled-coil domain of Dyb-1 [18]. Here, we refined this analysis by studyingadditionaldeletionsthatsubdividetheH2domain. Our results confirm the previously published data and show that H2 is involved in the interaction with Dys-1 in vitro. Within this domain, the first half seems to be particularly critical as deletions infringing on this side lead to a decrease in binding. Constructs 2¢5, 5¢2B and 5¢1 are of particular interest;thefirsttwoconstructsbreakataminoacid489and retain binding to Dys-1, whereas the third one breaks at position 487 and its binding affinity is reduced two-fold. Alternatively, this discrepancy might also be attributable to differences in the three dimensional structure of the helix imposed by amino acids on the C-terminal side of the breakpoint. The predicted three-dimensional structure of the various constructs has not been investigated so far. The two deletions of the H2 region that retained some in vitro binding activity (clones 2¢5 and 5¢2B) were unable to promote Dys-1–Dyb-1 interaction in the yeast two-hybrid system, which indicates thatthe yeast assay is more selective than the in vitro assay. A possible explanation may reside in the differences in protein concentrations. In the in vitro pull down experiment, the GST–Dys-1 moiety is in great excess toDyb-1,whereasDys-1andDyb-1arethoughttobeinthe same range of concentration in the yeast assay. In agree-ment with the yeast results, these two constructs (2¢5 and ... - tailieumienphi.vn