Heterologous expression of the human polybromo-1 protein in the methylotrophic yeast Pichia pastoris.

The human polybromo-1 protein (BAF180) is a known driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. BAF180 is the chromatin-targeting subunit of the PBAF complex. BAF180 has six bromodomains, two BAH domains, and one HMG box. Bromodomains are known to recognize acetylated-lysines on histones and play a role in nucleosome recognition. BAH domains are required for ubiquitination of PCNA, a key regulator of DNA damage. The putative HMG box, if functional, may be involved in DNA-binding. While the binding specificities of individual bromodomains have been studied by our lab and others, the results have failed to reach a consensus. The acetyl-histone binding features of the full-length protein is unknown and is the motivation for this work. The hypothetical HMG and BAH domains have not been studied and the actual function of these regions is currently unknown. Thus, the precise interactions of this large and complex protein are not well-studied. Advances in understanding this large protein have been hindered by the inability to express and purify recombinant full-length BAF180 protein. Currently, only phenomenological studies using BAF180 expressed in mammalian cells have been conducted. Here, we report the successful expression, purification of full-length biologically active BAF180 protein using the GAP promoter in the heterologous host Pichia pastoris. The ability to express full-length and mutated BAF180 will allow for biophysical binding studies. Knowledge of the binding interactions is critical for us to understand the role of BAF180 in cancer development and its progression.

The evolutionary genetics of highly divergent alleles of the mimicry locus in Papilio dardanus.

BACKGROUND: The phylogenetic history of genes underlying phenotypic diversity can offer insight into the evolutionary origin of adaptive traits. This is especially true where single genes have large phenotypic effects, for example in determining polymorphic mimicry in butterflies. Here, we characterise the evolutionary history of two candidate genes for the mimicry switch in the polymorphic Batesian mimic Papilio dardanus coding for the transcription factors engrailed and invected. RESULTS: We show that phased haplotypes associated with the dominant morphs f. poultoni and f. planemoides are phylogenetically highly divergent, in particular at non-synonymous sites. Some non-synonymous changes are shared between the divergent alleles suggesting either convergence or a shared ancestry. Gene trees for invected do not show this pattern. Despite their great divergence, all engrailed alleles of P. dardanus were monophyletic with respect to alleles of closely related species. Phylogenetic analyses therefore reveal no evidence for introgression from other species. A McDonald-Kreitman test conducted on a population sample from South Africa confirms a significant excess of intraspecific non-synonymous diversity in P. dardanus engrailed, suggesting long-term balanced polymorphism at this locus. CONCLUSIONS: The divergence between engrailed haplotypes suggests an evolutionary history distorted by selection with multiple changes reflecting recurrent selective sweeps. The high level of intraspecific polymorphism observed is characteristic of balancing selection on this locus, as expected if the gene engrailed is under phenotypic selection for the maintenance of multiple mimetic morphs. Non-synonymous changes in key functional portions of a major transcription factor are likely to be deleterious but if maintained in a dominant allele at low frequency, heterozygosity would reduce the associated genetic load.

Comparative genomics of the mimicry switch in Papilio dardanus.

The African Mocker Swallowtail, Papilio dardanus, is a textbook example in evolutionary genetics. Classical breeding experiments have shown that wing pattern variation in this polymorphic Batesian mimic is determined by the polyallelic H locus that controls a set of distinct mimetic phenotypes. Using bacterial artificial chromosome (BAC) sequencing, recombination analyses and comparative genomics, we show that H co-segregates with an interval of less than 500 kb that is collinear with two other Lepidoptera genomes and contains 24 genes, including the transcription factor genes engrailed (en) and invected (inv). H is located in a region of conserved gene order, which argues against any role for genomic translocations in the evolution of a hypothesized multi-gene mimicry locus. Natural populations of P. dardanus show significant associations of specific morphs with single nucleotide polymorphisms (SNPs), centred on en. In addition, SNP variation in the H region reveals evidence of non-neutral molecular evolution in the en gene alone. We find evidence for a duplication potentially driving physical constraints on recombination in the lamborni morph. Absence of perfect linkage disequilibrium between different genes in the other morphs suggests that H is limited to nucleotide positions in the regulatory and coding regions of en. Our results therefore support the hypothesis that a single gene underlies wing pattern variation in P. dardanus.

BAF180: Its Roles in DNA Repair and Consequences in Cancer.

In 2011, Varela et al. reported that the PBRM1 gene is mutated in approximately 40% of clear cell renal cell carcinoma cases. Since then, the number of studies relating PBRM1 mutations to cancers has substantially increased. BAF180 has now been linked to more than 30 types of cancers, including ccRCC, cholangiocarcinomas, esophageal squamous cell carcinoma, bladder cancer, and breast cancer. The mutations associated with BAF180 are most often truncations, which result in a loss of protein expression. This loss has been shown to adversely affect the expression of genes, likely because BAF180 is the chromatin recognition subunit of PBAF. In addition, BAF180 functions in numerous DNA repair mechanisms. Its roles in mediating DNA repair are likely the mechanism by which BAF180 acts a tumor suppressor protein. As research on this protein gains more interest, scientists will begin to piece together the complicated puzzle of the BAF180 protein and why its loss often results in cancer.

Characterising the phenotypic diversity of Papilio dardanus wing patterns using an extensive museum collection.

The history of 20th Century evolutionary biology can be followed through the study of mimetic butterflies. From the initial findings of discontinuous polymorphism through the debates regarding the evolution of mimicry and the step-size of evolutionary change, to the studies on supergene evolution and molecular characterisation of butterfly genomes, mimetic butterflies have been at the heart of evolutionary thought for over 100 years. During this time, few species have received as much attention and in-depth study as Papilio dardanus. To assist all aspects of mimicry research, we present a complete data-derived overview of the extent of polymorphism within this species. Using historical samples permanently held by the NHM London, we document the extent of phenotypic variation and characterise the diversity present in each of the subspecies and how it varies across Africa. We also demonstrate an association between "imperfect" mimetic forms and the transitional race formed in the area where Eastern and Western African populations meet around Lake Victoria. We present a novel portal for access to this collection, www.mimeticbutterflies.org, allowing remote access to this unique repository. It is hoped that this online resource can act as a nucleus for the sharing and dissemination of other collections databases and imagery connected with mimetic butterflies.

Serologic prevalence of MPV1 in mouse strains in a commercial laboratory mouse colony determined by using VP1 antigen.

A mouse parvovirus (designated MPV1f) was identified in a commercial laboratory mouse colony in Australia. The infection had not been detected by using an rNS1 parvovirus ELISA antigen even though the virus was genetically similar to other MPV1 variants reported previously. A recombinant biotinylated protein based on a truncated VP1 protein of the MPV1 strain was produced and used as antigen for ELISA and Western immunoblots to detect virus infection and determine the seroprevalence of infection in a colony of approximately 45,000 mice. Antibody-positive mice were detected in 8 of 11 rooms sampled, indicating that infection was widespread in the facility. Antibody was detected in 16.2% of 1161 sera obtained from 20 strains of mice. Seroprevalence varied among mouse strains, suggesting genetic variation in the susceptibility of mice to MPV1 or in their antibody response to infection, as has been reported previously in experimentally infected mice. Seroprevalence was high in some inbred strains, including DBA/2JArc and the random-bred strains Hsd:NIH and Arc:Arc(s). Antibody was not detected inC57BL/6J strains, and BALB/c strains showed low seroprevalence of MPV1f.

Strain- and age-associated variation in viral persistence and antibody response to mouse parvovirus 1 in experimentally infected mice.

The effect of mouse strain and age at infection on viral replication and concurrent antibody response to mouse parvovirus 1 (isolate MPV1f) was evaluated for 305 d after inoculation in 4 strains of mice. The results confirmed previous reports that mouse strain and age at infection are significant factors in viral persistence and antibody development and detection. Randombred Arc:Arc(s) mice originally bred from CD1 stock inoculated as juveniles (4 wk) or adults (8 wk) developed persistent viral infection for 152 d after inoculation and an antibody response that persisted for 295 d. Mice of C57BL/6J background inoculated as juveniles had detectable viral DNA in large intestinal content and tissues for 24 d after inoculation and an antibody response that persisted for 288 d. However, viral DNA was not detected in tissues of C57BL/6J mice inoculated as adults, although an antibody was detected for 111 d after inoculation; these results suggest probable viral replication in adult C57BL/6J mice but at levels below the limits of detection. BALB/cArc mice inoculated as juveniles or adults had detectable virus DNA in tissues for 108 to 242 d after inoculation, but no antibody was detected. Similarly, BALB/c-Foxn1(nu)/Arc mice had detectable levels of viral DNA in tissues for 98 to 131 d but no measurable antibody. The difficulty of detecting antibody in mice with a BALB/c background indicates they are unsuitable for routine surveillance of MPV1f infection.

HLS5, a novel RBCC (ring finger, B box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor.

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.