BIOLOGICAL OVERVIEW

The gene bric à brac (bab) is required for pattern formation along the proximal-distal axis of the leg and antenna of Drosophila (Godt, 1993). The French name bric à brac refers to the disorganized structure of the bab mutant ovaries. In bab mutant legs, the bristle pattern of the three central tarsal segments is transformed towards the pattern of the most proximal tarsal segment. bab function is dosage dependent and is required in a graded manner for the specification of tarsal segments. The graded requirement for bab correlates with its graded expression pattern, suggesting that the concentration of Bab protein specifies segment identity in the tarsus (Godt, 1993). In addition, bab mutant legs and antennae have segmentation defects. In addition, ovarian terminal filament formation depends on the Bab protein, which is expressed in the nuclei of terminal filament cells and is cell autonomously required (Godt, 1995). Disruption of terminal filament formation, together with defects of basal and interfollicular stalk development, leads to disruption of ovariole formation and female sterility in bab mutants. bab encodes a nuclear protein that contains a highly conserved BTB/POZ domain. The BTB/POZ protein-protein interaction domain is involved in homomeric and heteromeric associations with other BTB/POZ domains. Bab physically interacts with Bip2, a novel Drosophila TATA-box protein associated factor also termed (TAFII), also named dTAFII155. This interaction provides a direct link between BTB/POZ transcription factors and the basal transcriptional machinery (Pointud, 2001).

bab is required and expressed in a distinct proximal-distal domain of the limbs; the central region of the tarsus of the leg and the basal cylinder of the antenna. The domain of bab activity in limbs is apparently identical to the domain defined by the phenotype and expression pattern of the zinc finger transcription factor rotund (once mistakenly thought to be a RAS GTPase activator). In addition, this leg domain is characterized by the gene deadpan, which is expressed in a distal circumferential stripe in each of the segments TS1 to TS4. bab and rotund appear to act rather late in limb development, in contrast to genes that control the whole proximal-distal axis and appear to be required from embryogenesis onward, such as Distal-less and wingless. The subdivision of the tarsal primordium is a late event in the pattern formation of the leg and is also an evolutionarily recent step. Primitive insects only have one tarsal segment and the number of tarsal segments differs widely among more advanced insects. Taken together, this indicates that the bab/rotund domain is a distinct field for pattern formation during leg and antenna development (Godt, 1993).

Comparison of the bristle pattern in the tarsal segments of wild-type and bab mutant flies suggests that loss-of-function bab mutations cause a homeotic transformation of the three central tarsal segments (TS2-TS4) towards the basitarsus (TS1). This indicates a serial homology of segments in the tarsus. A similar conclusion was reached by Curt Stern in 1954, based on the finding of an extra sex comb on TS2 in extra sex comb-aristapedia double mutants. It may be assumed, therefore, that TS1-TS4 have the same basic pattern information that is modulated by bab activity in TS2-TS4 in order to give them a specification different from that of TS1. The data suggest a role for bab as a homeotic gene that is required along the proximal-distal axis of the legs to direct the developmental fate of TS2, TS3, and TS4 (Godt, 1993 and references therein).

The haploinsufficient transformation effect of bab alleles indicates that the specification of the tarsal segments depends on bab dosage. Also, the phenotypic series of bab alleles can be interpreted as a consequence of sequentially reduced levels of bab expression. The comparison of different bab alleles indicates a graded requirement for bab activity along the proximal-distal axis that becomes apparent in the higher sensitivity of TS2 compared to TS3 and TS4 transformation towards TS1. Given the dosage dependence, a simple explanation for this observation would be a graded distribution of the bab gene product along the proximal-distal axis. This explanation is corroborated by the different levels of bab expression that are observed in TS1- TS4. TS1 contains the lowest level of bab product and is considered the ground state. TS2, which is most sensitive to homeotic transformation to TS1, has a lower level of bab expression than TS3 and TS4. It is proposed, therefore, that the sensitivity to homeotic transformation towards TS1 is correlated with the concentration of the Bab protein in the different tarsal segments. It is unclear if this correlation holds for TS4, which contains equal or slightly lower levels of bab expression than TS3. In TS4, whose small size seems to allow the production of only one SCB, the usage of this morphological marker might not be sensitive enough to always detect a transformation to TS1 (Godt, 1993).

Is bab a morphogen? There are two possible explanations for how bab may act in a concentration-dependent manner for the specification of tarsal segments. bab may promote a binary decision between a TS1 fate and a non-TS1 fate, or bab may act as a morphogen and provide different positional values for the specification of TS2, TS3 and TS4. The latter possibility is supported by the graded distribution of the bab product. In addition, the analysis of metathoracic tarsal segments of weak and intermediate bab mutants shows a change in the bristle pattern of TS3 that can be interpreted as a transformation of TS3 towards TS2 rather than TS1. It is suggested, therefore, that bab may provide, depending on its concentration, different positional values for the specification of TS2 and TS3. It will be possible to assess the ability of the Bab protein to act as a morphogen by studies of bab overexpression and by examination of segment-specific molecular markers in bab mutants (Godt, 1993).

bab is the first gene for which a graded expression along the proximal-distal axis is described. The Bab protein distribution in the tarsal primordium reflects the distribution of the bab transcript, which indicates that the pattern of bab expression is regulated at the transcriptional level. The polar coordinate model proposes the formation of distal limb structures under the control of a circular coordinate which integrates anterior-posterior and dorsal-ventral positional values. For example, mutations in wg (a gene that encodes a secreted and diffusible protein and is believed to define circumferential positional values) have a drastic effect on the proximal-distal axis. However, it is not clear how genes like wg could induce a gradient of a nuclear protein along the proximal-distal axis. Whether Dll, which likely acts upstream of bab and is proposed to provide positional information along the proximal-distal axis, directly regulates bab expression is not known, but considering that the tarsus is a distinct Dll expression domain in the third larval instar, Dll might be involved in defining the domain and/or the pattern of bab expression (Godt, 1993).

The analysis of bab mutations indicates that bab has, in addition to its requirement for segment specification, a second function in limb development. bab mutations cause segmentation defects in the central region of the tarsus and in the homologous part of the antenna, the basal cylinder. Because strongly hypomorphic bab mutations cause more severe segmentation defects as hemizygotes than as homozygotes, it is assumed that amorphic bab mutations might lead to even stronger defects in segmentation (Pointud, 2001).

At the onset of metamorphosis, the bab product is distributed in a wave-like pattern in the tarsal primordium. Each tarsal fold shows a bell-shaped expression pattern of bab with the maximum level of expression at the ridge and the minimum in the furrow. Considering the segmentation defects in the tarsus of bab mutants, it is proposed that the wave-like pattern may be involved in the segmentation process of the tarsus. A number of theoretical models have utilized chemical wave patterns to explain how segmentation could occur. In these models, the waves compose a prepattern that reflect the structures that will develop, and the segment boundaries are proposed to be specified by the troughs or peaks of the wave pattern. Analysis of the molecular mechanisms of segmentation in the Drosophila embryo has shown that wave-like prepatterns are not part of the segmentation process. The finding that a wave-like pattern of bab exists in the developing tarsus suggests that segmentation in the tarsus occurs by a mechanism that differs from segmentation in the embryo (Godt, 1993).

In this context, it has to be asked whether the wave-like bab expression pattern can be considered a prepattern for segmentation of the tarsus. The wave-like pattern of the bab product at the onset of metamorphosis develops from a rather uniform distribution in the mid third larval instar. The wave-like pattern appears to form in parallel to the development of the tarsal folds. This, together with the observation that the segments of the tarsus seem to be already defined by the mid third larval instar, suggests that the wave-like distribution of the bab product is not likely to be a classical prepattern. In contrast to the embryo, however, pattern formation in the tarsal primordium occurs in a proliferating epithelium where a stable molecular prepattern is not expected. It has been suggested that new positional values in the growing imaginal disc are generated by intercalation between values that already exist. The development of the wave-like bab expression pattern may reflect this process. Early folding defects in the tarsal region of bab mutant imaginal discs indicate that bab is involved in the morphogenetic folding process. It is therefore proposed that in the growing field of the tarsal primordium, the morphogenetic process of segmentation and the development of the bab gene expression pattern may occur in a mutually dependent manner (Godt, 1993).

Sexually dimorphic abdominal pigmentation and segment morphology evolved recently in the melanogaster species group of the Drosophila. These traits are controlled by the bric-à-brac gene, which integrates regulatory inputs from the homeotic and sex-determination pathways. bab expression is modulated segment- and sex-specifically in sexually dimorphic species, but is uniform in sexually monomorphic species. It is suggested that bab has an ancestral homeotic function, and that regulatory changes at the bab locus played a key role in the evolution of sexual dimorphism. Pigmentation patterns specified by bab affect mating preferences, suggesting that sexual selection has contributed to the evolution of bab regulation (Kopp, 2000).

A key challenge in evolutionary biology is to identify genetic events responsible for morphological change, and to understand how changes at the molecular level affect development and translate into phenotypic diversity. To achieve this, two distinct approaches have been pursued in recent years: (1) comparative studies have revealed strong correlations between the expression patterns of individual regulatory genes during development and differences in morphology; (2) direct genetic analysis has been used to estimate the number and identity of genetic loci that contribute to morphological variation within and between species. Despite their respective successes, the two approaches remain far apart because of their different scales of analysis. Comparative studies have concentrated mainly on slowly evolving traits among high-level taxa, but genetic analyses are only possible among closely related species that produce viable and fertile hybrids (Kopp, 2000).

An approach to bridging this gap between evolutionary genetics and comparative embryology is to analyze and compare the development of rapidly evolving morphological traits. In many animals, secondary sexual characteristics evolve rapidly, making them good candidates for analysis. One such character in Drosophila is the pigmentation of adult abdominal segments. In D. melanogaster, abdominal pigmentation is sexually dimorphic. Segments 1 to 6 in females and 1 to 4 in males carry only a posterior stripe of dark pigment. However, segments 5 and 6 (A5 and A6) in males are completely pigmented, giving the species its name. This pattern is of recent evolutionary origin; in most Drosophila species, male-specific pigmentation is absent, so that females and males are pigmented identically. To understand how this new pattern originated and evolved, the regulatory circuit that controls its development has been characterized, and its operation has been compared in sexually dimorphic and monomorphic species (Kopp, 2000).

The development of sexually dimorphic external characteristics is controlled by the doublesex (dsx) gene. Alternative splicing of the dsx transcript produces a male-specific product in males (dsx^M), and a female-specific product in females (dsx^F). Loss of dsx function in females results in the development of male-like pigmentation, which can be suppressed by heat-shock dsx^F transgenes. Male-specific pigmentation is therefore expressed by default, and must be actively repressed by dsx^F (Kopp, 2000).

Thus, the development of sexually dimorphic pigmentation requires integration of homeotic and sex determination gene inputs. In investigating how this integration is achieved, a newly evolved genetic circuit has been discovered that appears to be responsible for the origin of male-specific pigmentation (Kopp, 2000).

A gene near the left tip of the third chromosome contributes to the variation in female abdominal pigmentation. In investigating this genetic region, it was found that loss of one copy of the bab locus results in the development of male-specific pigmentation in females, but has no effect on the male abdomen. Ectopic pigmentation in heterozygous bab females is suppressed by reducing the dosage of Abd-B, but is not eliminated by loss of omb. This suggests that bab+ represses the development of male-specific pigmentation in females by opposing the function of Abd-B. The bab locus contains two closely related genes, bab1 and bab2, which encode putative transcription factors with multiple roles in development. Ectopic pigmentation in females increases in the order bab1/+ < bab1/bab1 bab1bab2/+ bab1bab2/bab1, indicating that both genes are involved in repressing male pigmentation. For simplicity, the entire locus has been treated as one gene, bab, unless noted otherwise (Kopp, 2000).

The expression pattern of bab at the pupal stage when the adult epidermis develops reflects its sex- and segment-specific function. In females, bab expression is strongest in segments A2 and A3, and progressively weaker in A4, A5 and A6. In males, bab expression is considerably weaker than in females in all segments. Most strikingly, it is completely absent from A5 and A6. This pattern of bab repression correlates with the presence of sex-specific pigmentation in males, and its absence in females (Kopp, 2000).

To test whether bab+ is sufficient to repress pigmentation, the bab genes were ectopically expressed in the pupal abdomen. Low-level expression of bab+ results in the loss of male-specific pigmentation, but has no other effects on external morphology, indicating that differential regulation of bab plays a central role in establishing sexual dimorphism. bab+ can also repress non-sex-specific pigment stripes when expressed at a higher level. This suggests that bab+ acts as a general repressor of pigmentation, but that its effects are overridden by omb in the posterior part of each segment. Consistent with this, complete loss of both bab genes results in ectopic pigmentation of A2 to A7 in both sexes. This phenotype is not caused by expansion of Abd-B expression, which appears normal in these mutants. In bab homozygotes, the intensity of pigmentation is higher in the more posterior segments than in those more anterior. This suggests that pigmentation does not develop by default in the absence of bab, but is actively promoted by Abd-B and abd-A (Kopp, 2000).

The sexually dimorphic repression of bab in the posterior abdomen suggests that bab integrates the homeotic and sex determination regulatory inputs. To test this, bab expression was examined in Abd-B and dsx mutant backgrounds. Ectopic expression of Abd-B in A3 and A4 eliminates bab expression from these segments in males, and downregulates it in females. Conversely, bab is derepressed in A5-A7 in the mutants that lack Abd-B function in these segments. Together, these results indicate that bab expression in A5 and A6 is normally repressed by Abd-B. The slight downregulation of bab in A4 suggests that it is also weakly repressed by abd-A (Kopp, 2000).

In dsx-intersexes, bab is expressed in a male-like pattern, suggesting that dsx^F upregulates bab transcription in females. Abd-B and abd-A expression is identical in males, females and dsx -intersexes, indicating that bab is regulated independently by homeotic and sex-determination inputs. dsx^Dominant intersexes, which express both male- and female-specific dsx products, also show male-like expression of bab, indicating that dsx^M can interfere with dsx^F function. The two dsx isoforms encode transcription factors that bind the same DNA sequence, but have opposite effects on gene expression. dsx-intersexes differ from males in having a small unpigmented region at the anterior-lateral margin of A5, suggesting that dsx^M may have a slight negative influence on bab expression (Kopp, 2000).

These results suggest that bab+ regulates sexually dimorphic pigmentation by integrating regulatory inputs from the homeotic genes and the sex determination pathway. In this regulatory circuit, bab+ acts as a general repressor of pigmentation, and Abd-B and abd-A promote pigmentation in both sexes. In addition, Abd-B, and to a lesser extent abd-A, repress bab transcription. In males, this results in the absence of bab from A5 and A6, allowing Abd-B and abd-A to promote pigmentation in these segments. However, in females, dsx^F prevents bab transcription from being completely repressed by the homeotic genes. As a result, bab is present in A5 and A6 in females, where it blocks the ability of Abd-B and abd-A to promote pigmentation. In A2-A4, abd-A alone is not sufficient either to repress bab or to overcome its inhibitory effect on pigmentation; thus, only the omb-dependent striped pigmentation is generated. Because Abd-B, abd-A and dsx encode transcription factors, they may regulate bab expression directly (Kopp, 2000).

The central role of bab as an integrator of homeotic and sex-determination gene inputs suggests that changes in bab regulation may have been responsible for the evolution of sexually dimorphic pigmentation. In the subgenus Sophophora, male-specific pigmentation is present only in the melanogaster species group. Within this group, sexual dimorphism is seen in all species of the melanogaster subgroup and the closely related oriental subgroups, whereas the ananassae and montium subgroups contain both sexually dimorphic and sexually monomorphic species (Kopp, 2000).

In species with male-specific pigmentation of A5 and A6, bab expression is absent or strongly downregulated in these segments in males, but not in females. Moreover, in the sexually monomorphic species outside the melanogaster species group, bab expression is identical in both sexes and in all segments from A2 to A7. This correlation suggests that changes in the regulation of bab by Abd-B and dsx played an important role in the origin of sexually dimorphic pigmentation (Kopp, 2000).

bab+ regulates segment shape and bristle and trichome patterns in a manner reciprocal to Abd-B. Loss of bab+ function in females enhances posterior characteristics in A6, A7 and A8. No phenotype is seen in males, consistent with the absence of bab expression in posterior segments. Conversely, ectopic expression of bab transforms A6 and A7 to a more anterior identity in both males and females. These observations suggest that bab+ acts as an antagonist of Abd-B homeotic function, and that posterior abdominal characters are determined by the balance between Abd-B and bab activities (Kopp, 2000).

This model predicts that evolutionary changes in bab regulation should result in morphological transformation of Abd-B-expressing segments. Indeed, the entire suite of characteristics that distinguishes A5 and A6 from the more anterior segments in D. melanogaster is of recent evolutionary origin. In D. willistoni, bab is expressed strongly in A5 and A6 in males, whereas Abd-B is expressed in the same pattern as in D. melanogaster. As predicted, A5 and A6 are almost identical to the more anterior, non-Abd-B-expressing segments in the males of this species. In contrast, the melanogaster species group shows great diversity of bristle and trichome patterns in posterior abdominal segments. The two main lineages within this group show different patterns of evolution. In the clade composed of the melanogaster and oriental subgroups, male-specific pigmentation and bristle and trichome patterns have evolved in a concerted fashion. However, in the ananassae + montium lineage, these characteristics vary independently of each other, and sexually dimorphic bristle and trichome patterns are sometimes observed in species that do not show visible modulation of bab expression. This suggests that evolutionary changes have occurred not only in bab regulation, but also in the target genes of bab and in other genes regulated by Abd-B and dsx . Suppression of A7 development in males has occurred earlier in evolution than visible modulation of bab expression, despite the ability of bab to override this suppression (Kopp, 2000).

The rapid evolution of sexually dimorphic pigmentation and segment morphology may have been driven by sexual selection. Whether male-specific pigmentation confers a competitive advantage in D. melanogaster males was tested. Surprisingly, UAS-bab2 males, which lack male-specific pigmentation but are otherwise normal, enjoy the same mating success as wild-type males. Thus, although male pigmentation may have been important in the past, it appears to have little or no effect on female mating preferences in extant D. melanogaster (Kopp, 2000).

However, D. melanogaster males discriminate strongly against heterozygous bab females, which have ectopic male-specific pigmentation but are otherwise normal, compared with females with lightly pigmented A5 and A6. Importantly, white mutant males, which are visually impaired, mate equally with bab/+ and lightly pigmented females, suggesting that discrimination against bab heterozygous females is due to their pigmentation. These results suggest that female pigmentation is important in determining their attractiveness to males, and that the absence of male-specific pigmentation in females may be maintained by sexual selection (Kopp, 2000).

These findings indicate that changes in bab regulation have played an important part in the evolution of abdominal segment morphology. The presence of bab expression in all Drosophila species examined suggests that its roles in antagonizing the homeotic function of Abd-B and repressing pigmentation are ancestral. However, in the ancestral condition, bab expression was independent of Abd-B and dsx, resulting in sexually monomorphic pigmentation and segment morphology. In the melanogaster species group, bab evolved to be under the control of Abd-B and dsx. This eliminated bab from Abd-B-expressing segments in the male and resulted in a major transformation of male segment morphology. Subsequent diversification of pigmentation, bristle and trichome patterns was probably driven both by the fine-tuning of bab regulation and by changes in the downstream targets of bab and Abd-B (Kopp, 2000).

Two features of this genetic circuit make it highly plastic and evolvable: (1) the adult phenotype is sensitive to quantitative changes in bab expression; (2) the level of bab expression is determined by the balance between Abd-B and dsx^F inputs. If bab is regulated directly by Abd-B and dsx, then the evolution of sexually dimorphic pigmentation and segment morphology may ultimately be traced to the acquisition and modification of binding sites for the Abd-B and Dsx proteins in the cis-regulatory region of bab. Thus, even a subtle molecular change could be expressed phenotypically and become subject to selection (Kopp, 2000).

This evolutionary model is further supported by the presence of intraspecific genetic variation in sexually dimorphic pigmentation in many extant species. In at least one case, there is strong evidence that allelic differences at the bab locus contribute to this variation. Females found in natural populations of D. melanogaster vary widely in the extent of A6 pigmentation, ranging from near-zero to 100%. The locus with the largest effect on this variation has been mapped to the exact position of bab. These observations suggest that sexually dimorphic pigmentation evolved through fixation of intraspecific genetic variants at the bab locus (Kopp, 2000).

Fixation of new bab alleles was probably driven initially by 'runaway' sexual selection. In this case, a slight female preference for a weakly pronounced male character would initiate a positive feedback loop that would rapidly increase both the expression of the male character and the female preference for it. This self-reinforcing mechanism can drive rapid character divergence and create new species through sexual isolation. Male-specific pigmentation could evolve by this mechanism, with increasingly discriminating females selecting for increasingly dark males. However, once fixed, sexual characteristics can lose their significance as they are overtaken by newly evolving signals and as females become habituated and 'resistant' to old characters. This may explain the finding that male pigmentation has no effect on mating success in extant D. melanogaster (Kopp, 2000).

Whereas the runaway model explains the evolution of male sexual characters, it does not account for the absence of these characters in females, that is, sexual dimorphism. However, sexual dimorphism can be produced effectively by counter-selection against male-specific traits in females. Consistent with this, D. melanogaster males discriminate against females that have male-like pigmentation. In most Drosophila species, including D. melanogaster, males seek out females at feeding sites and attempt to court as many as possible. Courting other males is not only disadvantageous in competition for females, but may also carry a direct cost. Thus, males are probably selected for an ability to avoid courting other males, and pigmentation may be used to identify females at a distance (Kopp, 2000).

The evolution of bab regulation offers a tractable model of how selection creates new morphological characters through changes in DNA sequence. Analysis of the cis-regulatory elements of bab in sexually dimorphic and monomorphic species will help to clarify the molecular basis of morphological divergence between these taxa (Kopp, 2000).

GENE STRUCTURE

cDNA clone length - 4967

Bases in 5' UTR - 144

Exons - 2

Bases in 3' UTR - 1582

PROTEIN STRUCTURE

Amino Acids - 979

Structural Domains

The bab gene shares a homologous domain with the Drosophila genes tramtrack and Broad-Complex. Analysis of the 8 cDNAs isolated indicates that they are incomplete. Compilation of sequence data from the cDNAs and the genomic clones has allowed the identification of a 1.7 kb open reading frame that is adjacent to the P-lacZ insertion site. This open reading frame may not represent the entire Bab protein-coding region, and the analysis of additional cDNAs is required. However, it is believed that the sequences identified are part of the bab gene for three reasons.: (1) the P-lacZ insert of the bab^P allele is located immediately 3' of the identified open reading frame.; (2) the distribution of the detected RNA and protein is equivalent to the ß-gal expression pattern of the enhancer trap lines, and this expression pattern is consistent with the defects observed in the ovaries and limbs of bab mutants; (3) the anti-Bab r2 antibody, which is directed to a portion of the open reading frame, does not detect the Bab protein in the imaginal tissues of flies homozygous for the strong alleles bab^PR24 or bab^PRDS but does detect it in wild-type revertants of the bab^P allele (Godt, 1993).

Computer analysis of the 1.7 kb open reading frame has identified a strong similarity to two other Drosophila genes, tramtrack and Broad Complex. There is a 57% identity over 127 amino acid residues between Bab and Ttk; 59% over 112 amino acid residues between Bab and Br-C, and 55% over 113 amino acid residues between Ttk and Br-C. Both ttk and BR-C are genes that encode a family of zinc-finger proteins that are thought to be transcription factors. The highly conserved region (which has been named the BTB domain after BR-C, ttk and bab) is not part of the zinc-finger domain in the TTK and BR-C proteins, and its function is unknown (Godt, 1993).

bab was originally identified by the bab^P allele, a P-element insertion mutation that maps to polytene band 61F1-2. An open reading frame (ORF) adjacent to the bab^P insertion identified a BTB domain, a domain found primarily but not exclusively in Zn-finger containing transcription factors. In addition to bab, a second gene encoding a BTB domain (called BTB-II) was identified at 61F1-2 and found to have an expression pattern similar to bab. bab and BTB-II have been shown to constitute a gene complex. For clarity, the gene previously called bab will be renamed bab1, and BTB-II will be renamed bab2. The bab locus or bab will refer to both genes together (Couderc, 2002).

The bab1 and bab2 genes have the same orientation and show several structural similarities, suggesting that they are the result of a chromosomal duplication. bab1 and bab2 span approximately 60 kb and 25 kb of genomic DNA, respectively. The insertion point of bab^P is located in intron 1 of bab1, 236 bp downstream of the 5' splice site. Both genes have four introns, three of which are at homologous positions in the coding region. Sequence analysis of the bab1 and bab2 transcript predicts proteins of 967 and 1067 amino acids, respectively. Bab1 and Bab2 have two evolutionarily conserved domains in common. Outside of these domains Bab1 and Bab2 show only low sequence similarity to each other and no significant similarity to other proteins. A BTB domain, a conserved domain of 115 amino acids, is found in the N-terminal region of both proteins. In contrast to most known BTB domain-containing proteins that are transcriptional regulators, the BTB domains of Bab1 and Bab2 do not start within the first few amino acids of the predicted proteins but at amino acids 90 and 195, respectively. The Bab1 and Bab2 BTB domains are the most closely conserved domains within the BTB family (Couderc, 2002).

In contrast to many other known BTB domain-containing proteins that are transcription factors, neither Bab1 nor Bab2 contains a Zn-finger motif. However, these proteins have a second domain in common that is called BabCD for Bab-conserved domain. In both proteins, this domain is encoded by three exons with the splice sites at homologous positions. The BabCD contains two known motifs, a Psq domain and an AT-hook-like motif. The Psq domain can mediate DNA binding, and is named after pipsqueak (psq), another BTB domain-encoding gene. The Psq domain, which is 48 amino acids in length, is 97% identical between Bab1 and Bab2, and is the region of highest similarity within the BabCD. A Psq domain has also been identified in the Tkr (Tyrosine kinase-related) protein and in Piefke (CG15812). Piefke also has a BTB domain. The previously published sequence of Tkr lacks a BTB domain, however newly isolated cDNAs for the tkr gene show that it contains the BTB-III domain. Therefore, all five genes known to encode proteins with a Psq domain in Drosophila also have a BTB domain (Couderc, 2002).

The C-terminal region of the BabCD contains an AT-hook-like motif. It has the invariant peptide core motif R-G-R-P flanked on either side by other positively charged amino acids but does not correspond to any known AT-hook variant identified so far. AT-hook motifs have been shown to mediate binding to the minor groove of stretches of AT-rich DNA. Whether the AT-hook-like motif in Bab1 and Bab2 is involved in DNA-binding remains to be investigated. Both Bab proteins also have a short motif that is very rich in glutamine and histidine residues, which could aid transactivation. The domain architecture of Bab1 and Bab2, together with their nuclear localization, suggest that these proteins function as transcriptional regulators (Couderc, 2002).

date revised: 10 July 2002

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