RESUMO
In the studies reported here, we have examined the properties of the Mcp element from the Drosophila melanogaster bithorax complex (BX-C). We have found that sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and that they silence adjacent reporters by a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a "pairing-sensitive" silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Fatores de Transcrição , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Fushi Tarazu , Modelos Genéticos , Complexo Repressor Polycomb 1 , Sequências Reguladoras de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
Ethyl methanesulphonate (EMS) was used to induce 39 lethal and 13 karmoisin mutations within Df(3R)kar(3J), a nine-band deficiency extending from 87C1 to 87C9 (inclusive). Five complementation groups (four lethal and one visible) were identified and cytologically mapped between 87C4-5 and 87C9, one complementation group per band, with the exception of complementation group A, which is localized to 87C4-5. These positions were determined using a set of overlapping deficiencies, each having at least one breakpoint in the 87C1-9 region. Mutations within a single complementation group have similar lethal phases or subvital phenotypes, consistent with the notion that each complementation group represents a single functional locus. No mutations localized to 87C1-C3. The inability to induce mutations in the 87C1 heat-shock puff locus is consistent with the current interpretation of a duplication of coding sequences at the 87A7 and 87C1 heat-shock puffs.
RESUMO
Two antagonistic groups of genes, the trithorax- and the Polycomb-group, are proposed to maintain the appropriate active or inactive state of homeotic genes set up earlier by transiently expressed segmentation genes. Although some details about the mechanism of maintenance are available, it is still unclear how the initially active or inactive chromatin domains are recognized by either the trithorax-group or the Polycomb-group proteins. We describe an unusual dominant allele of a Polycomb-group gene, Enhancer of zeste, which mimics the phenotype of loss-of-function mutations in trithorax-group genes. This mutation, named E(z)(Trithorax mimic) [E(z)(Trm)], contains a single-amino-acid substitution in the conserved SET domain. The strong dominant trithorax-like phenotypes elicited by this E(z) allele suggest that the mutated arginine-741 plays a critical role in distinguishing between active and inactive chromatin domains of the homeotic gene complexes. We have examined the modification of E(z)(Trm) phenotypes by mutant alleles of PcG and trxG genes and other mutations that alter the phosphorylation of nuclear proteins, covalent modifications of histones, or histone dosage. These data implicate some trxG genes in transcriptional repression as well as activation and provide genetic evidence for involvement of histone modifications in PcG/trxG-dependent transcriptional regulation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Inativação Gênica , Proteínas de Insetos/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Fatores de Transcrição , Acetilação , Alelos , Sequência de Aminoácidos , Animais , Butiratos/farmacologia , Mapeamento Cromossômico , Cromossomos/ultraestrutura , Genes Dominantes , Heterozigoto , Histonas/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Mutação , Fenótipo , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 2 , Ligação Proteica , Estrutura Terciária de Proteína , Glândulas Salivares/ultraestrutura , Análise de Sequência de DNA , Transcrição GênicaRESUMO
RpII215(D) (50) (= D50) is a lethal mutation caused by the insertion of a 1.3-kb P element 5' to sequences encoding the largest (215 kilodaltons) subunit of Drosophila RNA polymerase II. In dysgenic males D50 reverted to nonlethality at frequencies ranging from 2.6 to 6.5%. These reversions resulted from loss of P element sequences. Genetic tests of function and restriction enzyme analysis of revertant DNAs revealed that 35% or more of the reversion events were imprecise excisions. Two meiotic mutations that perturb excision repair and postreplication repair (mei-9(a) and mei-41(D5), respectively) had no influence on reversion frequency but may have increased the proportion of imprecise excisions. We suggest that these excisions are by-products of, rather than intermediates in, the transposition process.
RESUMO
The region between 86F1,2 and 87B15 on chromosome 3 of Drosophila melanogaster, which contains about 27 polytene chromosome bands including the 87A7 heat-shock locus, has been screened for EMS-induced visible and lethal mutations. We have recovered 268 lethal mutations that fall into 25 complementation groups. Cytogenetic localization of the complementation groups by deficiency mapping is consistent with the notion that each band encodes a single genetic function. We have also screened for mutations at the 87A7 heat shock locus, using a chromosome that has only one copy of the gene encoding the 70,000 dalton heat-shock protein (hsp70). No lethal or visible mutations at 87A7 were identified from 10,719 mutagenized chromosomes, and no female-sterile mutations at 87A7 were recovered from the 1,520 chromosomes whose progeny were tested for female fertility. We found no evidence that a functional hsp70 gene is required for development under laboratory conditions.
Assuntos
Drosophila melanogaster/genética , Proteínas/genética , Animais , Deleção Cromossômica , Mapeamento Cromossômico , Genes , Ligação Genética , Proteínas de Choque Térmico , Temperatura Alta , MutaçãoRESUMO
The Abd-B gene, one of the three homeotic genes in the Drosophila bithorax complex (BX-C), is required for the proper identity of the fifth through the eighth abdominal segments (corresponding to parasegments 10-14) of the fruitfly. The morphological difference between these four segments is due to the differential expression of Abd-B, which is achieved by the action of the parasegment-specific cis-regulatory regions infra-abdominal-5 (iab-5), -6, -7 and -8. The dominant gain-of-function mutation Frontabdominal-7 (Fab-7) removes a boundary separating two of these cis-regulatory regions, iab-6 and iab-7. As a consequence of the Fab-7 deletion, the parasegment 12- (PS12-) specific iab-7 is ectopically activated in PS11. This results in the transformation of the sixth abdominal segment (A6) into the seventh (A7) in Fab-7 flies. Here we report that point mutations of the Abd-B gene in trans suppress the Fab-7 phenotype in a pairing-dependent manner and thus represent a type of transvection. We show that the observed suppression is the result of trans-regulation of the defective Abd-B gene by the ectopically activated iab-7. Unlike previously demonstrated cases of trans-regulation in the Abd-B locus, trans-suppression of Fab-7 is sensitive to heterozygosity for chromosomal rearrangements that disturb homologous pairing at the nearby Ubx locus. However, in contrast to Ubx, the transvection we observed in the Abd-B locus is insensitive to the allelic status of zeste. Analysis of different deletion alleles of Abd-B that enhance trans-regulation suggests that an extensive upstream region, different from the sequences required for transcription initiation, mediates interactions between the iab cis-regulatory regions and the proximal Abd-B promoter. Moreover, we find that the amount of DNA deleted in the upstream region is roughly proportional to the strength of trans-interaction, suggesting that this region consists of numerous discrete elements that cooperate in tethering the iab regulatory domains to Abd-B. Possible implications of the tethering complex for the regulation of Abd-B are discussed. In addition, we present evidence that the tenacity of trans-interactions in the Abd-B gene may vary, depending upon the tissue and stage of development.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Genes Homeobox , Genes de Insetos , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Abdome/embriologia , Abdome/inervação , Animais , Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/genética , Drosophila/embriologia , Masculino , Fenótipo , Mutação Puntual , Deleção de Sequência , Translocação GenéticaRESUMO
The suppressor of position effect variegation (PEV) locus Su-var(3)6 maps to 87B5-10. The breakpoints of deficiencies that define this interval have been placed on a 250-kb molecular map of the region. The locus is allelic to the ck19 complementation group previously shown to encode a type 1 serine-threonine protein phosphatase (PP1) catalytic subunit. When introduced into flies by P element-mediated transformation, a 5.8-kb genomic fragment carrying this gene overcomes the suppressor phenotype of Su-var(3)6(01) and recessive lethality of all mutations of the locus. Four of the mutant alleles at the locus show a broad correlation between high levels of suppression of PEV, a high frequency of aberrant mitosis and low PP1 activity in larval extracts. However, some alleles with low PP1 activity show weak suppression of PEV with a high frequency of abnormal mitosis, whereas others show strong suppression of PEV with normal mitosis. The basis for these discussed.
Assuntos
Drosophila melanogaster/genética , Mitose/genética , Mutação , Fosfoproteínas Fosfatases/genética , Animais , Northern Blotting , Mapeamento Cromossômico , Elementos de DNA Transponíveis , Feminino , Teste de Complementação Genética , Heterozigoto , Masculino , Mosaicismo , Proteína Fosfatase 1 , Proteínas Repressoras/genética , Transformação GenéticaRESUMO
The "enzyme-probe" method [Solti M, Friedrich P: Eur J Biochem 95:551, 1979] has been applied to characterize the cyclic AMP pool in wild-type Canton-S and memory-mutant dunceM11 strains of Drosophila melanogaster. The kinetics of cyclic AMP breakdown in whole fly homogenates by endogenous cyclic nucleotide phosphodiesterase(s) indicate that the cyclic AMP pool is divided into free and bound fractions. The bound fraction in Canton-S and dunceM11 is 0.5 and 1.5 pmole/mg fly, respectively. Considering the total cyclic AMP content of the two strains, 1.6 and 10 pmole/mg fly, respectively, we conclude that the bulk of excess cyclic AMP in the mutant is free nucleotide.
Assuntos
AMP Cíclico/metabolismo , Drosophila melanogaster/metabolismo , Animais , Compartimento Celular , Drosophila melanogaster/genética , Cinética , MutaçãoRESUMO
An extremely large cis-regulatory region generates the parasegment-specific expression patterns of the homeotic genes in the bithorax complex. We present evidence supporting the idea that this cis-regulatory region is subdivided into independent cis-regulatory domains. We describe a Ubx-lacZ transposon which is inserted into one of these domains, iab-7. The PS12-specific pattern of LacZ expression from this reporter indicates that it is subject to the control of the iab-7 cis-regulatory domain, but is protected from the effects of adjacent regulatory domains. Protection on the proximal side appears to be provided by the Fab-7 boundary element. Deletion of this boundary results in the ectopic activation of iab-7 in PS11 (where the iab-6 cis-regulatory domain normally functions). We show that the Fab-7 boundary, like other boundaries, has an unusual chromatin structure.
Assuntos
Elementos de DNA Transponíveis , Sequências Reguladoras de Ácido Nucleico , Animais , Cromatina/química , Drosophila melanogaster , Larva , Mutação , RNA Mensageiro/genética , Tórax/embriologia , beta-Galactosidase/genéticaRESUMO
The bithorax complex specifies the identity of parasegments 5-14 of Drosophila. Although nine parasegment-specific functions, abx/bx, bxd/pbx and iab-2 to iab-8,9 have been identified, the whole bithorax complex appears to encode only three classes of proteins, Ubx, abd-A and Abd-B. Many observations suggest that the parasegment-specific functions act as positive cis-regulatory elements of Ubx, abd-A and Abd-B. We report the molecular genetics of a new gain-of-function mutation, Fab-7, which transforms parasegment 11 into parasegment 12. Induction of Abd-B mutations in cis (one of which removes the Abd-B homeobox) causes reversion of the dominant phenotype, demonstrating that Fab-7 misregulates Abd-B. A 4 kb deletion, 30 kb downstream from the Abd-B transcription unit, is solely responsible for the Fab-7 phenotype. We consider that the parasegment-specific functions lie in DNA domains that are sequentially and independently 'opened' along the chromosome. Once a domain is opened, the cis-regulatory sequences within it can carry out their function. We propose that the Fab-7 deletion removes a boundary separating the iab-6 and iab-7 cis-regulatory regions (the functions specific for parasegments 11 and 12) allowing the open configuration of iab-6 to invade iab-7 in parasegment 11. This is strongly supported by our finding that Fab-7 can be caused to revert by lesions not only in iab-7 but also in iab-6.
Assuntos
Drosophila/genética , Regulação da Expressão Gênica , Genes Homeobox , Animais , Southern Blotting , Mapeamento Cromossômico , Cruzamentos Genéticos , DNA/análise , DNA/genética , Drosophila/anatomia & histologia , Drosophila/efeitos da radiação , Feminino , Genótipo , Larva , Masculino , MutaçãoRESUMO
Polytene chromosomes of good cytological quality from pseudonurse cells (PNCs) of fs(2)B and otu11 mutants were obtained, photomaps for otu11 mutants were constructed and the general characteristics of polytene chromosomes from salivary glands (SGs) and PNCs were compared. Three conditions were found to improve the cytological quality of PNC chromosomes: temperature below 18 degrees C, a protein-rich medium and presence of the Y-chromosome. Detailed comparison of the chromosome banding pattern from SGs and PNCs has shown only minor differences between them. The frequency of asynapsis appeared to be 10 times higher for PNC chromosomes. Despite previous reports, features such as breaks and ectopic contacts turned out to be also typical for PNC chromosomes, but with remarkably lower frequencies.
Assuntos
Cromossomos/ultraestrutura , Drosophila melanogaster/genética , Animais , Bandeamento Cromossômico , Cromossomos/genética , Feminino , Heterocromatina/genética , Heterocromatina/ultraestrutura , Microscopia de Contraste de Fase , Mutação/genética , Ovário/citologia , Ovário/ultraestrutura , Glândulas Salivares/ultraestruturaRESUMO
Little is known about the way higher-order chromatin structure influences gene expression and chromosome topology in general. Genetic analysis in Drosophila has led to the discovery of two classes of genes, the regulators of homeotic genes and the modifiers of position-effect variegation, which seem to be good candidates for encoding some of the factors regulating chromatin functions. The Trithorax-like gene we described here is required for the normal expression of the homeotic genes and is a modifier of position-effect variegation. We found that Trithorax-like encodes the GAGA factor which is involved in the formation of an accessible chromatin structure at promoter sequences. Our genetic analysis suggests that the chromatin modelling function of the GAGA factor is not restricted to promoter regions.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Proteínas de Homeodomínio , Fatores de Transcrição/genética , Animais , Cromatina/genética , Mapeamento Cromossômico , Elementos de DNA Transponíveis , Regulação da Expressão Gênica , Genes de Insetos , Masculino , Mutação , Regiões Promotoras Genéticas , Dedos de Zinco/genéticaRESUMO
A very large cis-regulatory region of approximately 300 kb is responsible for the complex patterns of expression of the three homeotic genes of the bithorax complex Ubx, abd-A and Abd-B. This region can be subdivided in nine parasegment-specific regulatory subunits. Recent genetic and molecular analysis has revealed the existence of two novel cis-regulatory elements Mcp and Fab-7. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B in parasegments 9 and 10. Similarly, Fab-7 is located between iab-6 and iab-7, the parasegment 11 and 12-specific regulatory units. Mcp and Fab-7 appear to function as domain boundaries that separate adjacent cis-regulatory units. We report the analysis of two new Mcp mutant deletions (McpH27 and McpB116) that allow us to localize sequences essential for boundary function to a approximately 0.4 kb DNA segment. These essential sequences closely coincide to a approximately 0.3 kb nuclease hypersensitive region in chromatin. We also show that sequences contributing to the Fab-7 boundary appear to be spread over a larger stretch of DNA, but like Mcp have an unusual chromatin structure.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes Homeobox , Proteínas de Homeodomínio , Hormônios de Inseto/genética , Proteínas Nucleares , Proteínas/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição , Animais , Sequência de Bases , Cromatina/química , Deleção de Genes , Dados de Sequência Molecular , Mutagênese , Análise de Sequência de DNA , TóraxRESUMO
Inappropriate expression of the Drosophila pair-rule gene, fushi tarazu (ftz), causes cuticular pattern deletions apparently complementary to those in ftz larvae. We show that the two patterns actually originate similarly, in both cases affecting the even-numbered parasegmental boundaries. The reciprocal cuticular patterns derive from differing patterns of selector gene expression (homoeotic transformations). The primary effect of ectopic ftz activity is to broaden ftz domains by autocatalytic activation of endogenous ftz expression in an additional anterior cell. This activates engrailed (en) and represses wingless (wg) expression, consistent with their proposed combinatorial control by ftz (and other pair-rule genes) to define parasegmental primordia. We propose that the anterior margin of each ftz stripe is normally defined by the posterior even-skipped (eve) boundary.
Assuntos
Regulação da Expressão Gênica , Fenótipo , Animais , Drosophila melanogaster , Temperatura Alta , Mutação , Supressão Genética , Transcrição GênicaRESUMO
Parasegmental (PS)-specific expression of the homeotic genes of the bithorax-complex (BX-C) appears to depend upon the subdivision of the complex into a series of functionally independent cis-regulatory domains. Fab-7 is a regulatory element that lies between iab-6 and iab-7 (the PS11- and PS12-specific cis-regulatory domains, respectively). Deletion of Fab-7 causes ectopic expression of iab-7 in PS11 (where normally only iab-6 is active). Two models have been proposed to account for the dominant Fab-7 phenotype. The first considers that Fab-7 functions as a boundary element that insulates iab-6 and iab-7. The second model envisages that Fab-7 contains a silencer element that keeps iab-7 repressed in parasegments anterior to PS12. Using a P-element inserted in the middle of the Fab-7 region (the bit transposon), we have generated an extensive collection of new Fab-7 mutations that allow us to subdivide Fab-7 into a boundary element and a Polycomb-respond element (PRE). The boundary lies within 1 kb of DNA on the proximal side of the bit transposon (towards iab-6). Deletions removing this element alone cause a complex gain- and loss-of-function phenotype in PS11; in some groups of cells, both iab-6 and iab-7 are active, while in others both iab-6 and iab-7 are inactive. Thus, deletion of the boundary allows activating as well as repressing activities to travel between iab-6 and iab-7. We also provide evidences that the boundary region contains an enhancer blocker element. The Polycomb-response element lies within 0.5 kb of DNA immediately distal to the boundary (towards iab-7). Deletions removing the PRE alone do not typically cause any visible phenotype as homozygotes. Interestingly, weak ectopic activation of iab-7 is observed in hemizygous PRE deletions, suggesting that the mechanisms that keep iab-7 repressed in the absence of this element may depend upon chromosome pairing. These results help to reconcile the previously contradictory models on Fab-7 function and to shed light on how a chromatin domain boundary and a nearby PRE concur in the setting up of the appropriate PS-specific expression of the Abd-B gene of the BX-C.
Assuntos
Cromatina/fisiologia , Proteínas de Drosophila , Drosophila/fisiologia , Genes de Insetos , Proteínas de Insetos/biossíntese , Abdome , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Elementos de DNA Transponíveis , Drosophila/embriologia , Drosophila/genética , Embrião não Mamífero/fisiologia , Elementos Facilitadores Genéticos , Deleção de Genes , Homozigoto , Proteínas de Insetos/genética , Mutação , Especificidade de Órgãos , Fenótipo , Complexo Repressor Polycomb 1 , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , TóraxRESUMO
The GAGA protein of Drosophila was first identified as a stimulatory factor in in vitro transcription assays using the engrailed and Ultrabithorax promoters. Subsequent studies have suggested that the GAGA factor promotes transcription by blocking the repressive effects of histones; moreover, it has been shown to function in chromatin remodeling, acting together with other factors in the formation of nuclease hypersensitive sites in vitro. The GAGA factor is encoded by the Trithorax-like locus and in the studies reported here we have used the maternal effect allele Trl13C to examine the functions of the protein during embryogenesis. We find that GAGA is required for the proper expression of a variety of developmental loci that contain GAGA binding sites in their upstream regulatory regions. The observed disruptions in gene expression are consistent with those expected for a factor involved in chromatin remodeling. In addition to facilitating gene expression, the GAGA factor appears to have a more global role in chromosome structure and function. This is suggested by the spectrum of nuclear cleavage cycle defects observed in Trl13C embryos. These defects include asynchrony in the cleavage cycles, failure in chromosome condensation, abnormal chromosome segregation and chromosome fragmentation. These defects are likely to be related to the association of the GAGA protein with heterochromatic satellite sequences which is observed throughout the cell cycle.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila/embriologia , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/fisiologia , Oogênese/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Animais , Sítios de Ligação , Ciclo Celular , Divisão Celular , Embrião não Mamífero/química , Feminino , Fatores de Transcrição Fushi Tarazu , Genes de Insetos , Genes Letais , Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/genética , Infertilidade Feminina , Hormônios de Inseto/análise , Mutação , Ovário/química , RNA Mensageiro/análise , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
The genetic and cytogenetic locations of the structural gene (Men) for malic enzyme have been determined. Men maps genetically between kar and ry at 51.73 +/- 0.02. Cytogenetically, Men probably lies in the proximal edge of 87D1,2, based on the results of mapping utilizing a number of deficiencies with breakpoints in that region. A number of null alleles have been recovered; heterozygotes for these nulls and a Men deficiency are both viable and fertile. These findings are related to the one band, one functional unit model of salivary gland chromosome structure.
Assuntos
Drosophila melanogaster/genética , Genes , Malato Desidrogenase/genética , Animais , Mapeamento Cromossômico , Cruzamentos Genéticos , Drosophila melanogaster/enzimologia , Feminino , Fertilidade , Heterozigoto , MasculinoRESUMO
Four dominant suppressor and one enhancer of variegation loci were mapped in the polytene chromosome region extending from section 86C to section 88B of the Drosophila melanogaster third chromosome using a set of deficiencies. The suppressor locus Su-var(3)14 maps in 86CD, Su-var(3)13 in 86F4-7, Su-var(3)6 in 87B4-7 and Su-var(3)7 in 87E4-5. The enhancer locus E-var(3)3 maps in 87E12-F11. Su-var(3)13, Su-var(3)6 and Su-var(3)7 are also defined by point mutant alleles originally identified by other criteria (Reuter et al. 1986). Duplications covering the suppressor loci Su-var(3)14, Su-var(3)13, Su-var(3)6 and Su-var(3)7 were found to reduce considerably the haplo-abnormal effect of heterozygous point mutants of the corresponding loci. One suppressor locus, Su-var(3)7, maps within a region which has previously been cloned. The positions of deficiency breakpoints delimiting the suppressor locus indicate that all the necessary sequences for its function are located within 10 kb of cloned DNA.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica , Animais , Mapeamento Cromossômico , Cromossomos/ultraestrutura , Clonagem Molecular , Teste de Complementação Genética , Supressão Genética , Transcrição GênicaRESUMO
The Drosophila bithorax complex Abdominal-B (Abd-B) gene specifies parasegmental identity at the posterior end of the fly. The specific pattern of Abd-B expression in each parasegment (PS) determines its identity and, in PS10-13, Abd-B expression is controlled by four parasegment-specific cis-regulatory domains, iab-5 to iab-8, respectively. In order to properly determine parasegmental identity, these four cis-regulatory domains must function autonomously during both the initiation and maintenance phases of BX-C regulation. The studies reported here demonstrate that the (centromere) distal end of iab-7 domain is delimited by the Fab-8 boundary. Initiators that specify PS12 identity are located on the proximal iab-7 side of Fab-8, while initiators that specify PS13 identity are located on the distal side of Fab-8, in iab-8. We use transgene assays to demonstrate that Fab-8 has enhancer blocking activity and that it can insulate reporter constructs from the regulatory action of the iab-7 and iab-8 initiators. We also show that the Fab-8 boundary defines the realm of action of a nearby iab-8 Polycomb Response Element, preventing this element from ectopically silencing the adjacent domain. Finally, we demonstrate that the insulating activity of the Fab-8 boundary in BX-C is absolutely essential for the proper specification of parasegmental identity by the iab-7 and iab-8 cis-regulatory domains. Fab-8 together with the previously identified Fab-7 boundary delimit the first genetically defined higher order domain in a multicellular eukaryote.