Sabyasachi Das

Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution.

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes

BackgroundHalophilic prokaryotes are adapted to thrive in extreme conditions of salinity. Identification and analysis of distinct macromolecular characteristics of halophiles provide insight into the factors responsible for their adaptation to high-salt environments. The current report presents an extensive and systematic comparative analysis of genome and proteome composition of halophilic and non-halophilic microorganisms, with a view to identify such macromolecular signatures of haloadaptation.ResultsComparative analysis of the genomes and proteomes of halophiles and non-halophiles reveals some common trends in halophiles that transcend the boundary of phylogenetic relationship and the genomic GC-content of the species. At the protein level, halophilic species are characterized by low hydrophobicity, over-representation of acidic residues, especially Asp, under-representation of Cys, lower propensities for helix formation and higher propensities for coil structure. At the DNA level, the dinucleotide abundance profiles of halophilic genomes bear some common characteristics, which are quite distinct from those of non-halophiles, and hence may be regarded as specific genomic signatures for salt-adaptation. The synonymous codon usage in halophiles also exhibits similar patterns regardless of their long-term evolutionary history.ConclusionThe generality of molecular signatures for environmental adaptation of extreme salt-loving organisms, demonstrated in the present study, advocates the convergent evolution of halophilic species towards specific genome and amino acid composition, irrespective of their varying GC-bias and widely disparate taxonomic positions. The adapted features of halophiles seem to be related to physical principles governing DNA and protein stability, in response to the extreme environmental conditions under which they thrive.

The evolution of adaptive immunity in vertebrates.

Approximately 500 million years ago, two types of recombinatorial adaptive immune systems (AISs) arose in vertebrates. The jawed vertebrates diversify their repertoire of immunoglobulin domain-based T and B cell antigen receptors mainly through the rearrangement of V(D)J gene segments and somatic hypermutation, but none of the fundamental AIS recognition elements in jawed vertebrates have been found in jawless vertebrates. Instead, the AIS of jawless vertebrates is based on variable lymphocyte receptors (VLRs) that are generated through recombinatorial usage of a large panel of highly diverse leucine-rich-repeat (LRR) sequences. Whereas the appearance of transposon-like, recombination-activating genes contributed uniquely to the origin of the AIS in jawed vertebrates, the use of activation-induced cytidine deaminase for receptor diversification is common to both the jawed and jawless vertebrates. Despite these differences in anticipatory receptor construction, the basic AIS design featuring two interactive T and B lymphocyte arms apparently evolved in an ancestor of jawed and jawless vertebrates within the context of preexisting innate immunity and has been maintained since as a consequence of powerful and enduring selection, most probably for pathogen defense purposes.

Evolutionary implications of a third lymphocyte lineage in lampreys

Jawed vertebrates (gnathostomes) and jawless vertebrates (cyclostomes) have different adaptive immune systems. Gnathostomes use T- and B-cell antigen receptors belonging to the immunoglobulin superfamily. Cyclostomes, the lampreys and hagfish, instead use leucine-rich repeat proteins to construct variable lymphocyte receptors (VLRs), two types of which, VLRA and VLRB, are reciprocally expressed by lymphocytes resembling gnathostome T and B cells. Here we define another lineage of T-cell-like lymphocytes that express the recently identified VLRC receptors. Both VLRC+ and VLRA+ lymphocytes express orthologues of genes that gnathostome γδ and αβ T cells use for their differentiation, undergo VLRC and VLRA assembly and repertoire diversification in the ‘thymoid’ gill region, and express their VLRs solely as cell-surface proteins. Our findings suggest that the genetic programmes for two primordial T-cell lineages and a prototypic B-cell lineage were already present in the last common vertebrate ancestor approximately 500 million years ago. We propose that functional specialization of distinct T-cell-like lineages was an ancient feature of a primordial immune system.

MicroRNA 125b inhibition of B cell differentiation in germinal centers

MicroRNAs 125a and 125b are predicted to be able to bind to the B lymphocyte-induced maturation protein-1 (BLIMP-1) and IFN regulatory protein-4 (IRF-4) transcription factors, which are essential for plasma cell differentiation. A computational survey of the human and mouse genomes revealed that miR-125a and miR-125b are members of a multigene family located in paralogous clusters. The miR-125a cluster on chromosome 19 in humans includes miR-99b and let-7e, whereas the miR-125b cluster on chromosome 21 includes miR-99a and miR-let-7c. Our analysis of the expression profiles for these six miRs during B lineage differentiation indicated that mature miR-125a, miR-125b, miR-99b and let-7e transcripts are preferentially expressed by the actively dividing centroblasts in germinal centers (GC). However, miR-99b and let-7e are not predicted to bind BLIMP-1 or IRF-4 transcripts, and binding to the untranslated region of BLIMP-1 and IRF-4 messenger RNAs could be confirmed only for miR-125b. When the effect of miR-125b over-expression on terminal B cell differentiation was evaluated in an LPS-responsive B cell line, the induction of BLIMP-1 expression and IgM secretion was inhibited in this model system. Furthermore, miR-125b over-expression inhibited the differentiation of primary B cells and compromised the survival of cultured myeloma cells. These findings suggest that miR-125b promotes B lymphocyte diversification in GC by inhibiting premature utilization of essential transcription factors for plasma cell differentiation.

Evolutionary dynamics of the immunoglobulin heavy chain variable region genes in vertebrates

Immunoglobulin heavy chains are polypeptides encoded by four genes: variable (IGHV), joining (IGHJ), diversity (IGHD), and constant (IGHC) region genes. The number of IGHV genes varies from species to species. To understand the evolution of the IGHV multigene family, we identified and analyzed the IGHV sequences from 16 vertebrate species. The results show that the numbers of functional and nonfunctional IGHV genes among different species are positively correlated. The number of IGHV genes is relatively stable in teleosts, but the intragenomic sequence variation is generally higher in teleosts than in tetrapods. The IGHV genes in tetrapods can be classified into three phylogenetic clans (I, II, and III). The clan III and/or II genes are relatively abundant, whereas clan I genes exist in small numbers or are absent in most species. The genomic organization of clan I, II, and III IGHV genes varies considerably among species, but the entire IGHV locus seems to be conserved in the subtelomeric or near-centromeric region of chromosome. The presence or absence of specific IGHV clan members and the lineage-specific expansion and contraction of IGHV genes indicate that the IGHV locus continues to evolve in a species-specific manner. Our results suggest that the evolution of IGHV multigene family is more complex than previously thought and that several factors may act synergistically for the development of antibody repertoire.

Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers

The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups (κ, λ, and σ isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess three IGL isotypes: κ, λ, and σ. Of these, two (κ and λ) are also found in reptiles and some mammals. The λ isotype is found in all tetrapods tested to date, whereas the κ isotype seems to have been lost at least in some birds and in the microbat. Conservation of the cladistic molecular markers suggests that they are associated with functional specialization of the three IGL isotypes. The genomic maps of IGL loci reveal multiple gene rearrangements that occurred in the evolution of tetrapod species. These rearrangements have resulted in interspecific variation of the genomic lengths of the IGL loci and the number and order of IGL constituent genes, but the overall organization of the IGL loci has not changed.

Definition of a third VLR gene in hagfish

Significance The jawless vertebrates (hagfish and lampreys) possess an alternative adaptive immune system in which variable lymphocyte receptors (VLRs) constructed of leucine-rich repeats are used to recognize foreign antigens. Three VLR genes have been identified in lampreys (VLRA, VLRB, and VLRC), but only two (VLRA and VLRB) have been found in hagfish. Here, we identified and characterized a third hagfish VLR gene. Our analysis indicates that the third hagfish VLR is the ortholog of lamprey VLRA, while the previously identified hagfish “VLRA” is the counterpart of lamprey VLRC. The demonstration of three orthologous VLR genes in hagfish and lampreys suggests that this anticipatory receptor system evolved in a common ancestor of the two jawless vertebrate lineages ∼480 Mya. Jawless vertebrates (cyclostomes) have an alternative adaptive immune system in which lymphocytes somatically diversify their variable lymphocyte receptors (VLR) through recombinatorial use of leucine-rich repeat cassettes during VLR gene assembly. Three types of these anticipatory receptors in lampreys (VLRA, VLRB, and VLRC) are expressed by separate lymphocyte lineages. However, only two VLR genes (VLRA and VLRB) have been found in hagfish. Here we have identified a third hagfish VLR, which undergoes somatic assembly to generate sufficient diversity to encode a large repertoire of anticipatory receptors. Sequence analysis, structural comparison, and phylogenetic analysis indicate that the unique hagfish VLR is the counterpart of lamprey VLRA and the previously identified hagfish “VLRA” is the lamprey VLRC counterpart. The demonstration of three orthologous VLR genes in both lampreys and hagfish suggests that this anticipatory receptor system evolved in a common ancestor of the two cyclostome lineages around 480 Mya.

Organization of lamprey variable lymphocyte receptor C locus and repertoire development.

Jawless vertebrates are pivotal representatives for studies of the evolution of adaptive immunity due to their unique position in chordate phylogeny. Lamprey and hagfish, the extant jawless vertebrates, have an alternative lymphocyte-based adaptive immune system that is based on somatically diversifying leucine-rich repeat (LRR)-based antigen receptors, termed variable lymphocyte receptors (VLRs). Lamprey T-like and B-like lymphocyte lineages have been shown to express VLRA and VLRB types of anticipatory receptors, respectively. An additional VLR type, termed VLRC, has recently been identified in arctic lamprey (Lethenteron camtschaticum), and our analysis indicates that VLRC sequences are well conserved in sea lamprey (Petromyzon marinus), L. camtschaticum, and European brook lamprey (Lampetra planeri). Genome sequences of P. marinus were analyzed to determine the organization of the VLRC-encoding locus. In addition to the incomplete germ-line VLRC gene, we have identified 182 flanking donor genomic sequences that could be used to complete the assembly of mature VLRC genes. Donor LRR cassettes were classifiable into five basic structural groups, the composition of which determines their order of use during VLRC assembly by virtue of sequence similarities to the incomplete germ-line gene and to one another. Bidirectional VLRC assembly was predicted by comparisons of mature VLRC genes with the sequences of donor LRR cassettes and verified by analysis of partially assembled intermediates. Biased and repetitive use of certain donor LRR cassettes was demonstrable in mature VLRCs. Our analysis provides insight into the unique molecular strategies used for VLRC gene assembly and repertoire diversification.

Distinct, ecotype-specific genome and proteome signatures in the marine cyanobacteria Prochlorococcus

BackgroundThe marine cyanobacterium Prochlorococcus marinus, having multiple ecotypes of distinct genotypic/phenotypic traits and being the first documented example of genome shrinkage in free-living organisms, offers an ideal system for studying niche-driven molecular micro-diversity in closely related microbes. The present study, through an extensive comparative analysis of various genomic/proteomic features of 6 high light (HL) and 6 low light (LL) adapted strains, makes an attempt to identify molecular determinants associated with their vertical niche partitioning.ResultsPronounced strand-specific asymmetry in synonymous codon usage is observed exclusively in LL strains. Distinct dinucleotide abundance profiles are exhibited by 2 LL strains with larger genomes and G+C-content ≈ 50% (group LLa), 4 LL strains having reduced genomes and G+C-content ≈ 35-37% (group LLb), and 6 HL strains. Taking into account the emergence of LLa, LLb and HL strains (based on 16S rRNA phylogeny), a gradual increase in average aromaticity, pI values and beta- & coil-forming propensities and a decrease in mean hydrophobicity, instability indices and helix-forming propensities of core proteins are observed. Greater variations in orthologous gene repertoire are found between LLa and LLb strains, while higher number of positively selected genes exist between LL and HL strains.ConclusionStrains of different Prochlorococcus groups are characterized by distinct compositional, physicochemical and structural traits that are not mere remnants of a continuous genetic drift, but are potential outcomes of a grand scheme of niche-oriented stepwise diversification, that might have driven them chronologically towards greater stability/fidelity and invoked upon them a special ability to inhabit diverse oceanic environments.