Madhusudan Choudhary

Evolutionary Analysis of the B56 Gene Family of PP2A Regulatory Subunits

Protein phosphatase 2A (PP2A) is an abundant serine/threonine phosphatase that functions as a tumor suppressor in numerous cell-cell signaling pathways, including Wnt, myc, and ras. The B56 subunit of PP2A regulates its activity, and is encoded by five genes in humans. B56 proteins share a central core domain, but have divergent amino- and carboxy-termini, which are thought to provide isoform specificity. We performed phylogenetic analyses to better understand the evolution of the B56 gene family. We found that B56 was present as a single gene in eukaryotes prior to the divergence of animals, fungi, protists, and plants, and that B56 gene duplication prior to the divergence of protostomes and deuterostomes led to the origin of two B56 subfamilies, B56αβε and B56γδ. Further duplications led to three B56αβε genes and two B56γδ in vertebrates. Several nonvertebrate B56 gene names are based on distinct vertebrate isoform names, and would best be renamed. B56 subfamily genes lack significant divergence within primitive chordates, but each became distinct in complex vertebrates. Two vertebrate lineages have undergone B56 gene loss, Xenopus and Aves. In Xenopus, B56δ function may be compensated for by an alternatively spliced transcript, B56δ/γ, encoding a B56δ-like amino-terminal region and a B56γ core.

Evolutionary constraints and expression analysis of gene duplications in Rhodobacter sphaeroides 2.4.1

BackgroundGene duplication is a major force that contributes to the evolution of new metabolic functions in all organisms. Rhodobacter sphaeroides 2.4.1 is a bacterium that displays a wide degree of metabolic versatility and genome complexity and therefore is a fitting model for the study of gene duplications in bacteria. A comprehensive analysis of 234 duplicate gene-pairs in R. sphaeroides was performed using structural constraint and expression analysis.ResultsThe results revealed that most gene-pairs in in-paralogs are maintained under negative selection (ω ≤ 0.3), but the strength of selection differed among in-paralog gene-pairs. Although in-paralogs located on different replicons are maintained under purifying selection, the duplicated genes distributed between the primary chromosome (CI) and the second chromosome (CII) are relatively less selectively constrained than the gene-pairs located within each chromosome. The mRNA expression patterns of duplicate gene-pairs were examined through microarray analysis of this organism grown under seven different growth conditions. Results revealed that ~62% of paralogs have similar expression patterns (cosine ≥ 0.90) over all of these growth conditions, while only ~7% of paralogs are very different in their expression patterns (cosine < 0.50).ConclusionsThe overall findings of the study suggest that only a small proportion of paralogs contribute to the metabolic diversity and the evolution of novel metabolic functions in R. sphaeroides. In addition, the lack of relationships between structural constraints and gene-pair expression suggests that patterns of gene-pair expression are likely associated with conservation or divergence of gene-pair promoter regions and other coregulation mechanisms.

The prevalence of gene duplications and their ancient origin in Rhodobacter sphaeroides 2.4.1

BackgroundRhodobacter sphaeroides 2.4.1 is a metabolically versatile organism that belongs to α-3 subdivision of Proteobacteria. The present study was to identify the extent, history, and role of gene duplications in R. sphaeroides 2.4.1, an organism that possesses two chromosomes.ResultsA protein similarity search (BLASTP) identified 1247 orfs (~29.4% of the total protein coding orfs) that are present in 2 or more copies, 37.5% (234 gene-pairs) of which exist in duplicate copies. The distribution of the duplicate gene-pairs in all Clusters of Orthologous Groups (COGs) differed significantly when compared to the COG distribution across the whole genome. Location plots revealed clusters of gene duplications that possessed the same COG classification. Phylogenetic analyses were performed to determine a tree topology predicting either a Type-A or Type-B phylogenetic relationship. A Type-A phylogenetic relationship shows that a copy of the protein-pair matches more with an ortholog from a species closely related to R. sphaeroides while a Type-B relationship predicts the highest match between both copies of the R. sphaeroides protein-pair. The results revealed that ~77% of the proteins exhibited a Type-A phylogenetic relationship demonstrating the ancient origin of these gene duplications. Additional analyses on three other strains of R. sphaeroides revealed varying levels of gene loss and retention in these strains. Also, analyses on common gene pairs among the four strains revealed that these genes experience similar functional constraints and undergo purifying selection.ConclusionsAlthough the results suggest that the level of gene duplication in organisms with complex genome structuring (more than one chromosome) seems to be not markedly different from that in organisms with only a single chromosome, these duplications may have aided in genome reorganization in this group of eubacteria prior to the formation of R. sphaeroides as gene duplications involved in specialized functions might have contributed to complex genomic development.

Use of the sucrose gradient method for bacterial cell cycle synchronization.

Although many undergraduate and graduate Cell and Molecular Biology courses study the bacterial cell cycle and the mechanisms that regulate prokaryotic cell division, few laboratory projects exist for the enhanced study of cell cycle characteristics in a standard teaching laboratory. One notable reason for this lack of engaging laboratory projects is, although bacterial cells can be grown fairly easily, these cultured cells are in a variety of cell cycle states. As such, to study and understand the factors that regulate bacterial cell division in morphological, physiological, and even molecular respects, it is necessary to have bacterial cells in the same stage of its cell cycle. This matching can be performed by a procedure called cell cycle synchronization.

Differential Selective Pressures Experienced by the Aurora Kinase Gene Family

Aurora kinases (AKs) are serine/threonine kinases that are essential for cell division. Humans have three AK genes: AKA, AKB, and AKC. AKA is required for centrosome assembly, centrosome separation, and bipolar spindle assembly, and its mutation leads to abnormal spindle morphology. AKB is required for the spindle checkpoint and proper cytokinesis, and mutations cause chromosome misalignment and cytokinesis failure. AKC is expressed in germ cells, and has a role in meiosis analogous to that of AKB in mitosis. Mutation of any of the three isoforms can lead to cancer. AK proteins possess divergent N- and C-termini and a conserved central catalytic domain. We examined the evolution of the AK gene family using an identity matrix and by building a phylogenetic tree. The data suggest that AKA is the vertebrate ancestral gene, and that AKB and AKC resulted from gene duplication in placental mammals. In a nonsynonymous/synonymous rate substitution analysis, we found that AKB experienced the strongest, and AKC the weakest, purifying selection. Both the N- and C-termini and regions within the kinase domain experienced differential selection among the AK isoforms. These differentially selected sequences may be important for species specificity and isoform specificity, and are therefore potential therapeutic targets.

A Nonsynonymous/Synonymous Substitution Analysis of the B56 Gene Family Aids in Understanding B56 Isoform Diversity.

Gene duplication leads to the formation of gene families, wherein purifying or neutral selection maintains the original gene function, while diversifying selection confers new functions onto duplicated genes. The B56 gene family is highly conserved; it is encoded by one gene in protists and fungi, and five genes in vertebrates. B56 regulates protein phosphatase 2A (PP2A), an abundant heterotrimeric serine/threonine phosphatase that functions as a tumor suppressor and consists of a scaffolding “A” and catalytic “C” subunit heterodimer bound to a regulatory “B” subunit. Individual regulatory B56 subunits confer disparate functions onto PP2A in various cell-cell signaling pathways. B56 proteins share a conserved central core domain, but have divergent N- and C-termini which play a role in isoform specificity. We carried out a nonsynonymous/synonymous substitution analysis to better understand the divergence of vertebrate B56 genes. When five B56 paralogs from ten vertebrate species were analyzed, the gene family displayed purifying selection; stronger purifying selection was revealed when individual B56 isoforms were analyzed separately. The B56 core experienced stronger purifying selection than the N- and C-termini, which correlates with the presence of several contacts between the core and the AC heterodimer. Indeed, the majority of the contact points that we analyzed between B56 and the AC heterodimer experienced strong purifying selection. B56 subfamilies showed distinct patterns of selection in their N- and C-termini. The C-terminus of the B56-1 subfamily and the N-terminus of the B56-2 subfamily exhibited strong purifying selection, suggesting that these termini carry out subfamily-specific functions, while the opposite termini exhibited diversifying selection and likely carry out isoform-specific functions. We also found reduced synonymous substitutions at the N- and C-termini when grouping B56 genes by species but not by isoform, suggesting species-specific codon bias may have a role in regulating B56 gene expression.

Evolution of Multipartite Genomes in Prokaryotes

Recent findings have shed light on the interplay and roles of multipartite genome structure in relation to bacterial survival and specialization. The majority of bacteria with two chromosomes are members of the Proteobacteria group and recent evidence suggests that the primary (CI) and the accessory chromosomes (CII) are essential and ancient partners of these complex prokaryotic genomes. However, accessory chromosomes have evolved more rapidly to provide increased metabolic plasticity as the CI encodes more essential proteins necessary for cell survival. The flexibility and the high divergence of CII may allow increased adaptability to specialized environments in which the possession of a single chromosome may not fully permit. Models and hypotheses pertaining to the formation of accessory chromosomes and the roles of different inherent genomic factors integral to the evolution of the accessory chromosomes in bacteria such as evolutionary constraints, horizontal gene transfer, partitioning of genes representing different COGs, gene regulation mechanisms, and replication mechanisms are discussed in this chapter.

Professional Practices in Undergraduate Research Programs.

The undergraduate research experience (URE) is an important avenue within a college trajectory in which students enhance their critical thinking, learn about the scientific process, and develop the knowledge and values that will guide their future scientific and professional careers. Individual institutions, programs, departments, and faculty administer undergraduate research differently, but each should adhere to a common set of guidelines which govern the research mentoring process. Adherence to standard practices will enhance the research experience for both students and mentors. This article examines standards and guidelines for professional practices involving undergraduate research and scholarship, and will discuss lapses and limitations that students and faculty frequently confront. The growth, support, and proper management of undergraduate research programs (URPs) at primarily undergraduate institutions (PUIs) is important for maintaining a talented pool of young scientists, as students benefit greatly from direct interactions with faculty mentors that predominate at PUIs.

Identification and Characterization of Replication Origins of MultipleChromosomes in Rhodobacter sphaeroides

DNA replication has been extensively studied in a number of bacterial species, which possess a unipartite genome consisting of a single circular chromosome. Approximately 10% of sequenced bacterial species have a multipartite genome structure, which is comprised of multiple chromosomes. However, the coordination and regulation of multi-chromosomal replication in bacteria remains poorly understood. Rhodobacter sphaeroides possesses a multipartite genome consisting of two chromosomes, the primary chromosome (CI) of approximately 3Mb and the secondary chromosome (CII) of 0.9 Mb. Z-curve and GC skew analyses revealed that CI and CII of R. sphaeroides exhibit three and five putative chromosomal origin regions, respectively. Then, the flanking regions of these putative regions were further analyzed in terms of gene conservation, gene density, and gene ratios between the corresponding forward and complement strands, previously identified near the biologically confirmed replicative origins of bacterial species that were closely related to R. sphaeroides. Subsequently, all the putative replicator regions were cloned into a pLO1 plasmid, a suicide vector in R. sphaeroides. The autonomous replication of these recombinant plasmids in R. sphaeroides was further examined using conjugation and molecular methods. Results demonstrated that CI and CII of R. sphaeroides have a single replication origin on its chromosomes, respectively, and this will provide the basis of future work on coordination and control of replication and segregation of multiple chromosomes in bacteria.