BIOLOGICAL OVERVIEW

scribbler (sbb) was characterized in four different laboratories. Yang (2000) showed that it is indispensable for the normal behavior of larvae. Naming the gene brakeless (bks), Senti (2000) and Rao (2000) showed that it is required for the axonal guidance of the Drosophila visual system, and as master of thickveins (mtv), Funakoshi (2001) showed that the gene shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Scribbler is a novel protein with a single putative zinc finger region and possesses a nuclear localization signal, suggesting that it is a nuclear protein. This overview will concentrate on the behavioral phenotype for sbb described in Yang's especially thorough genetic study, and the first analysis of the possible molecular function of sbb, as described in Funakoshi's 2001 study. The work of Senti and Rao is reviewed in the Effects of Mutation section.

The scribbler mutant was found by screening a collection of P-element-tagged second chromosome pupal lethal lines for abnormal foraging and/or locomotion behavior. One line (l(2)03432) carries a mutation that causes an unusual larval behavioral phenotype on nonnutritive agar. The scribbler larval trail covered a small area and looked as if the larva had scribbled on the agar surface. This was in direct contrast to the relatively straight trails of wild-type larvae. Scribbler behavior was quantitated in two ways. (1) The percentage of larvae in a line that exhibited scribbler behavior was calculated. (2) The number of 6-mm grid squares entered by each larva during its 5-min test on the agar substrate was counted. Scribbler behavior was 64% penetrant in homozygous sbb^l(2)03432 larvae. These larvae also entered significantly fewer grid squares than did their heterozygous sibs or larvae of the wild-type for^R (rover) control strain. Scribbler behavior is exhibited only when sbb^l(2)03432 mutant larvae are traveling on a nonnutritive substrate (agar) and not when mutant larvae forage on a yeast and water paste. Thus, the expression of scribbler turning behavior is conditional on the environment, specifically on the absence of food. The sbb^l(2)03432 mutation results in late-stage pupal lethality with 10% of the pupae emerging from the pupal case but dying within 3 days of emergence. These adult escapers lack part of a wing vein at the end of L5 (Yang, 2000).

In situ hybridization revealed that the sbb gene is expressed in the embryonic and larval CNS. The P[GAL4] system was used to manipulate gene expression to determine whether expression of the 3-kb SBB RNA in the nervous system alone is sufficient to rescue scribbler behavior. In this binary system the yeast transcription factor GAL4 directs the expression of any gene fused downstream of the activation sequence UAS, thus permitting ectopic expression of the fused gene. It was found that the expression of the UAS-sbb in the nervous system alone is sufficient to rescue scribbler behavior. When sbb was targeted to the nervous system alone, 60% of the expected number of flies emerged as fertile adults, indicating only partial rescue of the lethal phenotype. Targeted expression of the UAS-sbb to neurons does not rescue the L5 wing vein phenotype, suggesting that this phenotype results from sbb expression outside the nervous system, perhaps in the nonneuronal cells in the imaginal discs (Yang, 2000).

It is of interest that the scribbler turning phenotype is displayed only in the absence of food, since turning rate and localized traveling are known to be important components of food search behavior. Search behavior is the means by which most motile animals find essential resources and hence is a trait that can strongly influence the survival of an individual. Genetic control of search behavior is not unprecedented. It has been shown that larvae carrying the rover allele of the foraging (for) gene exhibit long foraging trails in a large yeast patch and tend to move between depleted food patches, whereas homozygous sitter larvae locate the closest food patch and remain feeding on it. Similarly, adult rover flies walk significantly farther from a recently consumed sucrose drop than sitter flies whose higher turning rate promotes revisiting and keeps the fly near the drop. sbb larvae exhibit characteristic patterns of food search behavior (turning, bending, and feeding movements); however, these patterns are exhibited solely in the absence of food (Yang, 2000 and references therein).

Many developmental genes are known to be pleiotropic; their gene products play multiple roles throughout development. Genes that affect both larval and adult behavior can play important roles in development and sometimes have vital functions (for example, in Drosophila, learning genes such as dunce, latheo, and linotte; courtship mutants such as fruitless and fickle; larval behavioral mutants such as foraging and tamas; and ion channel mutants such as slow poke). One well-studied example of a pleiotropic gene that affects behavior is the learning gene dunce (dnc). Mutations in dnc cause female sterility and abnormal learning. The dnc gene is widely expressed; its expression is not restricted to the mushroom bodies of the fly brain known to play an important role in learning and memory. Pleiotropic genes that play a role in behavior are usually expressed in multiple tissues and during more than one developmental stage. Mutations in pleiotropic genes often affect more than one aspect of neuronal structure and/or function (Yang, 2000 and references therein).

sbb is a pleiotropic gene with a vital function. Four out of five (sbb^l(2)03432, sbb^l(2)04440, sbb^l(2)k00702, and sbb^l(2)04525 but not sbb^EP(2)0328) P-element insertion alleles of sbb exhibit lethality, primarily in the late pupal stage. Mutations in the sbb gene lead to multiple phenotypic defects that include larval scribbler behavior, pupal lethality, and a defect in the pattern of the L5 wing vein in the adult escapers. sbb transcripts are observed in all developmental stages and in multiple tissue types (the embryonic and larval CNS and the imaginal discs). These expression data support the phenotypic data suggesting that sbb likely functions during multiple developmental stages and in more than one tissue. The four different-sized transcripts suggest the existence of at least four different Scribbler isoforms that may arise from differential RNA splicing or alternative polyadenylation or initiation. It is predicted that some of these isoforms will have different functions and will be found in different tissues. Some initial support for this prediction comes from the finding of a body-specific transcript (7.8 kb) and from the lack of complete rescue of pupal lethality when sbb was only targeted to neuronal cells. Further studies are needed to address the question of how, when, and where sbb acts to accomplish its pleiotropic functions (Yang, 2000).

One of the pleiotropic functions of sbb alluded to by Yang (2000) is an effect of wing morphogenesis. This function has been addressed by Funakoshi (2001), who shows that sbb shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Drosophila wings are patterned by a morphogen, Decapentaplegic, a member of the TGFbeta superfamily, that is expressed along the anterior and posterior compartment boundary. The distribution and activity of Dpp signaling is controlled in part by the level of expression of its major type I receptor, thickveins (tkv). The level of tkv is dynamically regulated by Engrailed and Hedgehog. sbb, termed master of thickveins (mtv) by Funakoshi, downregulates expression of tkv in response to Hh and En. mtv expression is controlled by En and Hh, and is complementary to tkv expression. mtv integrates the activities of En and Hh that shape tkv expression pattern. Thus, mtv plays a key part of regulatory mechanism that makes the activity gradient of the Dpp morphogen (Funakoshi, 2001).

Dpp signaling activity can be visualized by using the antibody against phosphorylated Mothers against dpp (p-Mad): the distribution of the Dpp morphogen activity largely depends on the levels of the Tkv receptor. tkv expression, which is monitored by expression of beta-galactosidase in the tkv-lacZ enhancer trap line, is downregulated by Hh along the A/P border where dpp expression is induced by the same signal. The basal level of tkv is higher in the P compartment than it is in the A compartment. This complex pattern appears to shape the activity gradient of Dpp directly. The p-Mad level is low along the A/P border where tkv is downregulated. The gradient of the p-Mad distribution is steeper in the P compartment than it is in the A compartment, probably because high levels of Tkv limit the movement of Dpp; since the spread of Dpp would be less in the P compartment, its gradient of activity would be expected to be steeper (Funakoshi, 2001).

mtv was identified by characterizing the enhancer trap lines, 1E1 and l(2)k00702, that generate expression patterns largely complementary to that of tkv in wing discs except at the dorsoventral compartment border in the peripheral region, where both genes are expressed at high levels. Distribution of the transcript revealed by in situ hybridization with a probe prepared from the corresponding cDNA is consistent with the pattern of the enhancer trap lines. Only the longer form of bks/sbb/mtv mRNA is predominantly detected in imaginal discs (Funakoshi, 2001).

In order to know whether mtv has a role in regulating tkv, a deletion mutant allele, mtv⁶, was made by imprecise excision of the P-element and used for clonal analysis. It is believed that mtv⁶ is a strong hypomorphic allele, because its transcript can only encode a 19 amino acid polypeptide, which lacks most of putative functional domains of the Mtv protein. In mtv⁶ clones, tkv-lacZ levels are autonomously upregulated indicating that Mtv represses tkv. When a large mtv clone is induced in the area including the A/P border, tkv-lacZ levels become uniform within the clone, suggesting that mtv plays an important role in regulating a dynamic pattern of tkv expression throughout the wing pouch. p-Mad levels are also upregulated in a graded manner. This is consistent with the fact that tkv is derepressed within mtv mutant clones because ectopically induced Tkv upregulates p-Mad levels. No significant changes in dpp transcription levels were observed within mtv mutant clones, thus it is concluded that mtv shapes p-Mad spatial distribution through regulation of Tkv levels (Funakoshi, 2001).

To address whether the increased p-Mad levels produced in mtv clones result in the activation of targets of the Dpp signaling pathway, the expression of spalt (sal) and optomotor blind (omb) were studied. Within the mtv clones, expression of those marker genes is upregulated as expected in both A and P compartments. When clones are located at the edge of the sal-expressing domain, a slight expansion of sal expression is observed, as in the case of p-Mad staining. However, when clones are located at a distance from the sal-expressing domain, no upregulation is observed, presumably because in this location, Dpp signaling activity is below the required threshold for activity (Funakoshi, 2001).

Adult phenotypes of the mtv clones were examined and it was found that they are reminiscent of those caused by overexpression of tkv. When the mtv clone is anterior to the second vein, an ectopic vein is generated anterior to the second vein, probably due to the expansion of the sal expression domain, and triple-row bristles are transformed to double-row bristles. This suggests that the elevated tkv levels in mtv clones cause these cells to differentiate as if they are adjacent to the A/P compartment boundary. Another clone shows a modest overproliferation of the tissue: this is also consistent with the notion that the mtv mutation elevates the level of the Dpp signal, as has been reported by overexpression of Mad. The clone at the A/P border also disrupts the vein pattern. The upregulation of wild-type tkv can generate identical phenotypes to those observed in mtv clones: upregulation of sal expression in the wing pouch, bifurcation of veins and transformation of bristles in the adult wing. Thus, it is concluded that the major target of the mtv activity that organizes the A/P pattern is tkv (Funakoshi, 2001).

The fact that tkv expression is repressed by hh at the A/P border and mtv is highly expressed in the same cells has prompted an examination of whether mtv mediates hh dependent tkv repression. tkv-lacZ levels were examined in clones of cells mutant for patched (ptc), which encodes the Hh receptor. Hh signal transduction is constitutively active in the absence of ptc activity. Anterior compartment ptc clones cause cell-autonomous repression of tkv. In the clones of cells mutant both for mtv and ptc, however, tkv levels are elevated as in the mtv singly mutant clones. This indicates that mtv mediates hh-dependent tkv regulation along the A/P border. mtv-lacZ expression was monitored within clones of cells mutant for smoothened (smo), which encodes a component of the Hh receptor complex and is required for Hh signaling. Within the clones located at the A/P border, mtv-lacZ expression is repressed, indicating that Hh signaling induces the high level of mtv expression along the A/P border. This was also confirmed by the fact that mtv-lacZ levels are elevated within pka mutant clones, in which Hh signaling is constitutively active. These results show that Hh represses tkv levels by upregulating its negative regulator, mtv (Funakoshi, 2001).

As described earlier, the basal tkv level in the P compartment is higher than it is in the A compartment and it is responsible for the asymmetric structure of the wing. The mtv expression pattern is complementary to the tkv expression pattern. The possibility that mtv is also responsible for regulating the basal level of tkv expression was also examined. Initially, it was asked whether the level of tkv is regulated by en in the P compartment. Within clones of cells mutant for en in the P compartment, the tkv-lacZ level is lower than that seen in the A compartment, suggesting that tkv expression is regulated by en. This is confirmed by the observation that, within clones of cells ectopically expressing en in the A compartment, the tkv level is elevated in comparison to that seen in the P compartment in an autonomous way. Within en and mtv double mutant clones, tkv transcription levels are derepressed as in mtv single mutant clones, indicating that mtv mediates en dependent tkv regulation. Ectopically expressed en downregulates mtv-lacZ in the A compartment, implying that low levels of mtv in the P compartment are under the control of en regulation. These results altogether indicate that en regulates the high basal level of tkv expression in the P compartment by downregulating mtv expression (Funakoshi, 2001).

It is concluded that the central region of the wing is patterned by Hh but not by Dpp although Dpp expression is induced by Hh in this region. This is because Dpp signaling is downregulated by Hh by upregulating mtv, which causes repression of tkv expression. The patterning by Hh appears to be ensured by lowering the Dpp signaling, which would otherwise interfere with the Hh morphogen activity, because upregulation of Dpp signaling by overexpressing tkv or by eliminating mtv activity can alter the vein patterns there. Therefore, the mtv-dependent tkv regulation is required both for Hh and Dpp morphogen activities. The patterning along the A/P border between veins 3 and 4 may be more complicated. The dorsal mtv mutant clone at the A/P border disrupts the vein pattern; vein 3 is displaced posteriorly, which is similar to the phenotype associated with sal mutant clones. The fact that mtv is downregulated in sal mutant clones might explain the phenotype if Mtv has a role in mediating Sal activity, which positions vein 3 through regulating target genes such as the iroquois gene complex. Further analysis is required to elucidate whether tkv function is linear or parallel to this regulatory cascade (Funakoshi, 2001).

The mechanism that shapes the tkv pattern and hence the Dpp morphogen gradient is unique in that it makes the mtv expression pattern that is made by integrating En and Hh signals complementary to the tkv pattern. Thus, the mtv expression pattern acts as a 'negative' for generating the tkv pattern. en positive cells initiate the cascade of the patterning along the A/P axis by expressing Hh, which both acts as short-range morphogen and induces the long-range morphogen, Dpp. Here it is proposed that not only does En induce expression of the morphogen, but it also shapes the morphogen activity gradient by regulating its receptor level via Mtv (Funakoshi, 2001).

Drosophila Brakeless interacts with Atrophin and is required for tailless-mediated transcriptional repression in early embryos

Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. Mutations were isolated in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, an expanded expression pattern was noted of the Krüppel (Kr) and knirps (kni) genes. Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. Both Drosophila and human Brakeless and Atrophin interact in vitro, and it is proposed that they act together as a co-repressor complex in many developmental contexts. The possibility is discussed that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA) (Haecker, 2007).

Repression plays a pivotal role in establishing correct gene expression patterns that is necessary for cell fate specification during embryo development. For example, in the early Drosophila embryo, repression by gap and pair-rule proteins is essential for specifying the positions of the 14 segments of the animal. The mechanisms by which transcriptional repressors delimit gene expression borders are not well understood. However, many repressors require co-repressors for function. In the Drosophila embryo, the CtBP and Groucho co-repressors are required for activity of many repressors. Atrophin has been identified as a co-repressor for Even-skipped and Tll. Still, co-regulators for several important transcription factors in the early embryo have not yet been identified. Therefore a screen was performed for novel maternal factors that are required for establishing correct gene expression patterns in the early embryo (Haecker, 2007).

From this screen, mutations were identified in the bks gene that cause severe phenotypes on gap gene expression and embryo segmentation. The Bks protein is evolutionarily conserved between insects and deuterostomes, but has not been characterized in any species except Drosophila, in which it has been shown to repress runt expression in photoreceptor cells and thickveins expression in wing imaginal discs. However, the molecular function of Bks has been unknown. This study shows that Bks interacts with the transcriptional repressor Tll, is recruited to target gene CRMs, and will repress transcription when targeted to DNA (Haecker, 2007).

Tll has been shown to utilize Atrophin as a co-repressor. Atrophin genetically interacts with Tll and physically interacts with its ligand binding domain. Atrophin binding is conserved in nuclear receptors within the same subfamily, such as Seven-Up in Drosophila as well as Tlx and COUP-TF in mammals. When expressed in mammalian cells, Drosophila Atrophin and mouse Atrophin-2 interact with the histone deacetylases HDAC1 and HDAC2. Histone deacetylation may therefore be part of the mechanism by which Atrophin functions as a co-repressor. Another recent report described genetic interactions among bks and atrophin mutants in the formation of interocellar bristles in adult flies. Furthermore, it was shown that atrophin mutants have virtually identical phenotypes as bks mutants, including de-repression of runt expression in the eye, thickveins expression in the wing, and Kr and kni expression in the embryo (Haecker, 2007).

Both proteins are recruited to the kni CRM, a Tll-regulated target gene, in the embryo. Importantly, Atrophin and Bks interact in vitro and that they can be co-immunoprecipitated from S2 cells. It is proposed that Bks and Atrophin function together as a co-repressor complex, and based on the similar bks and atrophin mutant phenotypes at several developmental stages, the complex may function throughout development. These results are compatible with the existence of a tripartite complex consisting of Tll, Bks, and Atrophin. Bks binding to Tll is enhanced by the Tll DNA binding domain, whereas the interaction of Tll with Atrophin is mediated through the C-terminal ligand binding domain. Tll may therefore simultaneously interact with Bks and Atrophin. Alternatively, Tll interacts separately with Bks and Atrophin on the kni CRM. In either case, both Bks and Atrophin are required for full Tll activity. However, at high enough Tll concentration, Bks activity is dispensable. Some bks embryos misexpressing Tll still repress kni expression, and overexpressing Tll from a heat-shock promoter can repress the posterior kni stripe in both wt and bks mutant embryos. For this reason, it is believed that Bks and Atrophin are cooperating as Tll co-repressors, so that Tll function is only partially impaired by the absence of either one. It was found that Tet-Bks-mediated repression in cells is insensitive to the deacetylase inhibitor trichostatin A (TSA). It is possible, therefore, that whereas Atrophin-mediated repression may involve histone deacetylation, Bks could repress transcription through a separate mechanism (Haecker, 2007).

These results have not revealed any differences between the molecular functions of the two Bks isoforms. Both Bks-A and Bks-B repress transcription when tethered to DNA, and the sequences that mediated binding to Tll and Atrophin are shared between the two isoforms. However, the bks³³⁹ allele that selectively affects the Bks-B isoform causes a weaker, but comparable phenotype to the stronger bks alleles that disrupt both isoforms. Therefore, the C-terminus of Bks-B provides a function that is indispensable for embryo development and regulation of kni expression. This part of Bks-B contains two regions (D3 and D4) that are highly conserved in insects and loosely conserved in deuterostome Bks sequences, but does not resemble any sequence with known function. The only sequence similarity to domains found in other proteins is a single zinc-finger motif in Bks-B. Preliminary results indicate that the zinc finger in isolation or together with the conserved D2 domain does not exhibit sequence-specific DNA binding activity. Indeed, multiple zinc fingers are generally required to achieve DNA binding specificity. Instead, Bks is likely brought to DNA through interactions with Tll and other transcription factors (Haecker, 2007).

Atrophins are required for embryo development in C. elegans, Drosophila, zebrafish, and mice. In vertebrates, two atrophin genes are present. Atrophin-1 is dispensable for embryonic development in mice, and lacks the N-terminal MTA-2 homologous domain that interacts with histone deacetylases . However, the homologous C-termini of Atrophin-1 and Atrophin-2 can interact, and it was found that this domain can also bind to the human Bks homolog ZNF608. Atrophin-1 interacts with another co-repressor-associated protein as well, ETO/MTG8, and can repress transcription when tethered to DNA. These data are consistent with the emerging view that deregulated transcription may be an important mechanism for the pathogenesis of polyglutamine diseases. Recent evidence indicates that interactions with the normal binding partners may cause toxicity of polyglutamine-expanded proteins such as Ataxin-1 . It will be interesting to investigate whether the interaction between human Bks homologs and Atrophin-1 is important for the neuronal toxicity of polyglutamine-expanded Atrophin-1 (Haecker, 2007).

GENE STRUCTURE

Sequence analysis of the 3.6-kb and 10.5-kb transcripts revealed that they differ in both their start and termination sites. Their deduced amino acid sequences are identical at the N-terminal end. The 10.5-kb transcript, when compared to the 3.6-kb transcript, encodes an additional 1373 amino acids at the C-terminal end (Rao, 2000).

A total of 13 bks/sbb cDNA clones were analyzed in detail and were found to fall into two classes. The longest clones in each class are 3.1 kb and 8.1 kb in length, with complete open reading frames encoding proteins of 929 and 2302 amino acids respectively. These two cDNA classes are referred to as bksA and bksB respectively. Comparison of the cDNA and genomic sequences indicates that the longer bksB transcript arises from alternative splicing at a donor site that overlaps the stop codon of bksA, leading to the addition of further 3' coding exons in bksB. In situ hybridization experiments performed with probes prepared from either the common region of bksA and bksB, or the unique region of bksB, shows that bks transcripts are uniformly and ubiquitously expressed in the eye imaginal disc (Senti, 2000).

PROTEIN STRUCTURE

Amino Acids - 929 and 2310

Structural Domains

DNA sequencing reveals that the 3.6-kb sbb transcript contains a long open reading frame encoding 929 amino acids. This 929-amino-acid protein contains 2 putative cAMP/cGMP-dependent protein kinase phosphorylation sites, 15 protein kinase C phosphorylation sites, and 7 casein kinase II phosphorylation sites. A PSORT (prediction of protein sorting signals and localization sites in amino acid sequences) search reveals two nuclear localization signals at position 840 (PPAKRVK) and position 841 (PAKRVKH) with 94.1% reliability. This suggests that the Scribbler protein is a nuclear protein. No homology was found with any known protein in the database when a BLASTP search of this deduced amino acid sequence was performed. However, a BLASTN search did reveal four EST clones isolated from mouse heart (AI644380, AI614389), Xenopus neurula (AI031376), and human (AI223051) that are highly homologous (70–90% identity) with the C-terminal region of the Scribbler protein. These data suggest the existence of Scribbler counterparts in vertebrates (Yang, 2000).

The 10.5-kb transcript contains a larger open reading frame (encoding 2302 amino acids) than the 3.6-kb transcript. The first 929 amino acids encoded by the 10.5-kb transcript are the same as the protein encoded by the 3.6-kb ORF. This larger protein contains one single zinc finger (C2H2 type) domain and one tyrosine kinase phosphorylation site that are not part of the first 929 amino acids shared with the 3.6-kb transcript. The PSORT search reveals five nuclear localization signals in this larger 2302-amino-acid protein. A BLASTP search of this deduced amino acid sequence shows homology with a human EST clone (AB002293) that contains a single zinc finger motif that has 70% identity within 30 amino acids in the zinc finger region. This human EST clone was originally isolated from a male brain cDNA library. This zinc finger region of the 2302 amino acids is also highly homologous (63% identity) with a Zebrafish EST clone (AI722347) suggesting the existence of a conservative single zinc finger domain (Yang, 2000).

Both the 3.6-kb and 10.5-kb gene products are novel proteins that are most likely localized within the nucleus. The large form contains one zinc finger C2H2 type motif that is different from the zinc finger domains found in numerous nucleic acid-binding proteins that have two or more sets of zinc finger motifs. In this case, each set of zinc finger motifs interacts with the same DNA sequence with similar affinities. For example one peptide thought to function in metal transport and/or modulation of gene expression has been found to contain one single zinc finger domain. Other experimental evidence has shown that zinc finger domains can play a role in protein-protein interactions. It is hypothesized that the larger Scribbler protein encoded by the 10.5-kb transcript may play a role in nucleic acid-binding and/or protein-protein interactions (Yang, 2000).

Sequence analysis of the bks cDNA reveals a single long ORF corresponding to a protein of 929 amino acids. The predicted molecular weight is 88 kDa. Bks protein does not resemble any protein of known function, nor does it contain known protein domains, motifs, or any significant stretches of sequence homology to known proteins. Bks protein contains an unusually high content of Ser (23.7%) and Thr (7.6%); repeated stretches of Ser-rich sequences are found in the amino-terminal half of the polypeptide. Sequences related to Bks, however, have been identified in public databases of ESTs from human (GenBank accession no. AI223051), mouse (accession nos. AI644380 and AI614389), and Xenopus (accession no. AI031376). These ESTs may represent either portions of bks homologs or proteins that contain an evolutionarily conserved domain with undefined function (Rao, 2000).

The two Bks proteins have a most unusual structure, with long stretches of low-complexity sequence. Both are also unusually hydrophilic. BksA has a particularly high content of serine (24.0%) and glycine (14.0%) residues, and polar residues in general (DEGKQRST, 67.3%). Hydrophobic residues (LVIFM) make up only 10.8% of BksA, whereas 99% of the proteins in SWISS-PROT have a hydrophobic amino acid content in excess of 13.0%. The unique region of BksB has a somewhat different composition, but also of low complexity, with proline (14.3%), glutamine (13.5%) and glycine (11.4%) as the most abundant residues. Scattered amongst these low-complexity regions are several small islands of high complexity. One of these, unique to BksB (residues 1112-1137) could be reliably identified as a classical C2H2 zinc finger domain. This region also shows high homology to predicted proteins from human, mouse, zebrafish and frog EST sequences and yeast and pufferfish genomic sequences. For the pufferfish genomic sequence, homology extends beyond the predicted zinc finger domain to include additional high-complexity sequences in the common region at the C terminus of BksA and continuing into the unique region of BksB (residues 869-968). Other ESTs also show homology to this region, which includes an almost perfectly conserved 63 amino acid motif in predicted pufferfish and mouse proteins. Remarkably, this motif is not found in any other Drosophila protein, and is not encoded at all within the C. elegans genome. In addition to these highly conserved motifs, another high-complexity region near the C terminus of BksB (residues 1986-2094) also shows significant but somewhat lower homology to a predicted human protein. These conserved high-complexity regions are likely to represent important functional domains present in Bks proteins (Senti, 2000).

date revised: 15 January 2001

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