Biology subjects

Kulkarni, A.

Publications and source records attributed to Kulkarni, A..

82 records · Page 5Linked to original sources

bioRxiv2019

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. The machinery was tested in two mosquito species. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least10 days post-delivery. In Anopheles gambiae, vitellogenin gene was silenced by Vg-crRNA injection two hours post-blood meal, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the - and {delta}-subunits of COPI genes were silenced by a post-blood meal crRNA injection, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. This study adds a programmable CRISPR tool to manipulate RNA in mosquitoes.

molecular biology↗

bioRxiv2019

Evidence of multifaceted functions of codon usage in translation within the model beetle Tribolium castaneum

Synonymous codon use is non-random. Codons most used in highly transcribed genes, often called optimal codons, typically have high gene counts of matching tRNA genes (tRNA abundance) and promote accurate and/or efficient translation. Non-optimal codons, those least used in highly expressed genes, may also affect translation. In multicellular organisms, codon optimality may vary among tissues. At present however, codon use remains poorly understood in multicellular organisms. Here, we studied codon usage of genes highly transcribed in germ line (testis, ovary) and somatic tissues (gonadectomized males and females) of the beetle Tribolium castaneum. The results demonstrate that: 1) the majority of optimal codons were organism-wide, the same in all tissues, and had numerous matching tRNA gene copies (Opt-codon{uparrow}tRNAs), consistent with translational selection; 2) some optimal codons varied among tissues, suggesting tissue-specific tRNA populations; 3) wobble tRNA were required for translation of certain optimal codons (Opt-codonwobble), possibly allowing precise translation and/or protein folding; and 4) remarkably, some non-optimal codons had abundant tRNA genes (Nonopt-codon{uparrow}tRNAs), and genes using those codons were tightly linked to ribosomal and stress-response functions. Thus, Nonopt-codon{uparrow}tRNAs codons may regulate translation of specific genes. Together, the evidence suggests that codon use and tRNA genes regulate multiple translational processes in T. castaneum.

evolutionary biology↗

bioRxiv2019

Absence of a faster-X effect in beetles (Tribolium, Coleoptera)

BackgroundThe faster-X effect, namely the rapid evolution of protein-coding genes on the X-chromosome, has been reported in numerous metazoans. However, the prevalence of this phenomenon across metazoans and its potential causes remain largely unresolved. Analysis of sex-biased genes may elucidate its possible mechanisms: a more pronounced faster-X effect in male-biased genes than in female-biased or unbiased genes, suggests fixation of recessive beneficial mutations rather than genetic drift. Further, theory predicts that the faster-X effect should be promoted by X-chromosome dosage compensation, but this topic remains rarely empirically examined.\n\nResultsHere, we asked whether we could detect a faster-X effect in genes of the beetle Tribolium castaneum (and T. freemani orthologs), which has X/Y sex-determination and heterogametic males. Our comparison of protein sequence divergence (dN/dS) on the X-chromosome versus autosomes indicated the complete absence of a faster-X effect. Further, analyses of sex-biased gene expression revealed that the X-chromosome was strongly enriched for ovary-biased genes, which evolved under exceptionally high constraint. An evaluation of male X-chromosome dosage compensation in the gonads and in non-gonadal somatic tissues showed an extreme lack of compensation in the testis. This under-expression of the X chromosome in males may limit the phenotypic effect, and therefore likelihood of fixation, of recessive beneficial X-linked mutations in genes transcribed in male gonads.\n\nConclusionsWe show that these beetles display a rare unequivocal example of the absence of a faster-X effect in a metazoan. We propose two potential causes for this, namely high constraint on X-linked ovary-biased genes, and an extreme lack of dosage compensation of genes transcribed in the testis.

evolutionary biology↗

bioRxiv2019

Cortical Foxp2 supports behavioral flexibility and developmental dopamine D1 receptor expression

Genetic studies have associated FOXP2 variation with speech and language disorders and other neurodevelopmental disorders involving pathology of the cortex. In this brain region, FoxP2 is expressed from development into adulthood, but little is known about its downstream molecular and behavioral functions. Here, we characterized cortex-specific Foxp2 conditional knockout mice and found a major deficit in reversal learning, a form of behavioral flexibility. In contrast, they showed normal activity levels, anxiety, and vocalizations, save for a slight decrease in neonatal call loudness. These behavioral phenotypes were accompanied by decreased cortical dopamine D1 receptor (D1R) expression at neonatal and adult stages, while general cortical development remained unaffected. Finally, using single-cell transcriptomics, we identified at least five excitatory and three inhibitory D1R-expressing cell types in neonatal frontal cortex, and we found changes in D1R cell type composition and gene expression upon cortical Foxp2 deletion. Strikingly, these alterations included non-cell-autonomous changes in upper-layer neurons and interneurons. Together these data support a role for Foxp2 in the development of dopamine-modulated cortical circuits and behaviors relevant to neurodevelopmental disorders.

neuroscience↗

bioRxiv2019

Single-cell analysis of Foxp1-driven mechanisms essential for striatal development

The striatum is a critical forebrain structure for integrating cognitive, sensory, and motor information from diverse brain regions into meaningful behavioral output. However, the transcriptional mechanisms that underlie striatal development and organization at single-cell resolution remain unknown. Here, we show that Foxp1, a transcription factor strongly linked to autism and intellectual disability, regulates organizational features of striatal circuitry in a cell-type-dependent fashion. Using single-cell RNA-sequencing, we examine the cellular diversity of the early postnatal striatum and find that cell-type-specific deletion of Foxp1 in striatal projection neurons alters the cellular composition and neurochemical architecture of the striatum. Importantly, using this approach, we identify the non-cell autonomous effects produced by disrupting Foxp1 in one cell-type and the molecular compensation that occurs in other populations. Finally, we identify Foxp1-regulated target genes within distinct cell-types and connect these molecular changes to functional and behavioral deficits relevant to phenotypes described in patients with FOXP1 loss-of-function mutations. These data reveal cell-type-specific transcriptional mechanisms underlying distinct features of striatal circuitry and identify Foxp1 as a key regulator of striatal development.

neuroscience↗

bioRxiv2019

CO2 conversion to methane and biomass in obligate methylotrophic methanogens in marine sediments

Methyl substrates are important compounds for methanogenesis in marine sediments but diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. Here, we demonstrate that RNA-stable isotope probing (SIP) requires 13C-labeled bicarbonate as co-substrate for identification of methylotrophic methanogens in sediment samples of the Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens incorporate 60 to 86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably more than what can be predicted from known metabolic pathways ([~]40% contribution). In slurry experiments amended with the marine methylotroph Methanococcoides methylutens, up to 12% of methane was produced from CO2, indicating that CO2-dependent methanogenesis is an alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens grow in fact mixotrophically on methyl compounds and DIC. Thus, the observed high DIC incorporation into lipds is likely linked to CO2-dependent methanogenesis, which was triggered when methane production rates were low. Since methylotrophic methanogenesis rates are much lower in marine sediments than under optimal conditions in pure culture, CO2 conversion to methane is an important but previously overlooked methanogenic process in sediments for methylotrophic methanogens.

microbiology↗

bioRxiv2018

Serial synapse formation through filopodial competition for synaptic seeding factors

Following axon pathfinding, growth cones transition from stochastic filopodial exploration to the formation of a limited number of synapses. How the interplay of filopodia and synapse assembly ensures robust connectivity in the brain has remained a challenging problem. Here, we developed a new 4D analysis method for filopodial dynamics and a data-driven computational model of synapse formation for R7 photoreceptor axons in developing Drosophila brains. Our live data support a serial synapse formation model, where at any time point only a single synaptogenic filopodium suppresses the synaptic competence of other filopodia through competition for synaptic seeding factors. Loss of the synaptic seeding factors Syd-1 and Liprin- leads to a loss of this suppression, filopodial destabilization and reduced synapse formation, which is sufficient to cause the destabilization of entire axon terminals. Our model provides a filopodial winner-takes-all mechanism that ensures the formation of an appropriate number of synapses.

neuroscience↗

bioRxiv2018

Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida, USA

The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but sporadically detected in Ae. aegypti. In this study, we used a two-step PCR assay to detect Wolbachia in wild-collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A Loop-mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild specimens of Ae. aegypti from various cities in New Mexico and in 2/46 (4.3%) wild specimens of Ae. aegypti from St. Augustine, Florida. We did not detect Wolbachia in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico in 2018.

ecology↗

bioRxiv2018

TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) is powerful technique to study transcriptional regulation. However, the requirement of millions of cells to generate results with high signal-to-noise ratio precludes it in the study of small cell populations. Here, we present a Tagmentation-Assisted Fragmentation ChIP (TAF-ChIP) and sequencing method to generate high-quality results from low cell numbers. The data obtained from the TAF-ChIP approach is amenable to standard tools for ChIP-Seq analysis, owing to its high signal-to-noise ratio. The epigenetic profiles from TAF-ChIP approach showed high agreement with conventional ChIP-Seq datasets, thereby underlining the utility of this approach.

genomics↗

bioRxiv2018

Molecular evolution of DNMT1 in vertebrates: duplications in marsupials followed by positive selection

DNA methylation is mediated by a conserved family of DNA methyltransferases (Dnmts). The human genome encodes five Dnmts: Dnmt1, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L. Despite their high degree of conservation among different species, genes encoding Dnmts have been duplicated and/or lost in multiple lineages throughout evolution, indicating that the DNA methylation machinery has some potential to undergo evolutionary change. However, little is known about the extent to which this machinery, or the methylome, varies among vertebrates. Here, we study the molecular evolution of Dnmt1, the enzyme responsible for maintenance of DNA methylation patterns after replication, in 79 vertebrate species. Our analyses show that all studied species exhibit a single copy of DNMT1, with the exception of tilapia and marsupials (tammar wallaby, koala, Tasmanian devil and opossum), each of which exhibits two apparently functional DNMT1 copies. Our phylogenetic analyses indicate that DNMT1 duplicated before the divergence of marsupials (i.e., at least ~75 million years ago), thus giving rise to two DNMT1 copies in marsupials (copy 1 and copy 2). In the opossum lineage, copy 2 was lost, and copy 1 recently duplicated again, generating three DNMT1 copies: two putatively functional genes (copy 1a and 1b) and one pseudogene (copy 1{psi}). Both marsupial copies (DNMT1 copies 1 and 2) are under purifying selection, and copy 2 exhibits elevated rates of evolution and signatures of positive selection, suggesting a scenario of neofunctionalization. This gene duplication might have resulted in modifications in marsupial methylomes and their dynamics.

evolutionary biology↗