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Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes.

Biedler JK, Tu Z - BMC Evol. Biol. (2010)

Bottom Line: Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus.In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae.AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Fralin Biotech Center, Blacksburg, VA 24061, USA.

ABSTRACT

Background: The maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in Drosophila melanogaster and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as bottleneck are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito Aedes aegypti to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a Medea gene drive system.

Results: Two novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1) in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the AaKLC2.1 start codon shows promoter activity at least as early as 3 hours in the developing Ae. aegypti embryo. The AaKLC2.1 promoter activity reached ~1600 fold over the negative control at 5 hr after egg deposition.

Conclusions: Transcriptome profiling by use of high throughput sequencing technologies has proven to be a valuable method for the identification and discovery of early and transient zygotic genes. The evolutionary investigation of the KLC gene family reveals that duplication is a source for the evolution of new genes that play a role in the dynamic process of early embryonic development. AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.

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Coding sequence structure and domains of kinesin light chain genes of mosquitoes and D. melanogaster. On the left are the AaKLC2-like genes and on the right are the AaKLC1-like genes. Vertical bars show location of introns and intron size is given below bars. Note conservation of gene structure (vertical lines show conservation of intron position). Intron sizes are shown as number of bases and are given below vertical bars. Rab5-bind, Rabaptin 5-binding domain. TPR, tetratricopeptide repeat domain. Shaded parts indicate those genes that have been analyzed by RT-PCR and Illumina transcriptome profiling (only AaKLC2.1 is shown). Predicted introns are taken from Vectorbase.org and Ensembl.org.
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Figure 1: Coding sequence structure and domains of kinesin light chain genes of mosquitoes and D. melanogaster. On the left are the AaKLC2-like genes and on the right are the AaKLC1-like genes. Vertical bars show location of introns and intron size is given below bars. Note conservation of gene structure (vertical lines show conservation of intron position). Intron sizes are shown as number of bases and are given below vertical bars. Rab5-bind, Rabaptin 5-binding domain. TPR, tetratricopeptide repeat domain. Shaded parts indicate those genes that have been analyzed by RT-PCR and Illumina transcriptome profiling (only AaKLC2.1 is shown). Predicted introns are taken from Vectorbase.org and Ensembl.org.

Mentions: Structural analysis supports the categorization of two KLC gene groups, the AaKLC1-like and the AaKLC2-like genes (Figure 1). AaKLC2.1 is an intronless gene with a predicted open reading frame (ORF) of 1419 nucleotides (nt). AaKLC2.2 is also intronless and has 98% nt and aa identity to AaKLC2.1 in its coding region. The lack of introns in the AaKLC2-like genes is notable since it has been reported that 70% of Drosophila early zygotic genes do not have introns presumably for increased efficiency of transcription with fast-cycling nuclei [1]. A likely ortholog of the AaKLC2 genes was identified in C. quinquefasciatus, which is an intronless gene having a similar ORF length (gene ID: CPIJ002971). However, no similar genes were found in the genome sequences of An. gambiae [15], An. stephensi (8× coverage genome assembly, Tu unpublished), D. melanogaster, or other insects. Thus from genome analysis, it appears that AaKLC2-like genes are restricted to mosquitoes of the subfamily Culicinae. The expression of the C. quinquefasciatus gene will need to be verified by RT-PCR or other methods to determine if it is indeed an early and transient zygotic gene. This is why we have not called it CqKLC2.


Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes.

Biedler JK, Tu Z - BMC Evol. Biol. (2010)

Coding sequence structure and domains of kinesin light chain genes of mosquitoes and D. melanogaster. On the left are the AaKLC2-like genes and on the right are the AaKLC1-like genes. Vertical bars show location of introns and intron size is given below bars. Note conservation of gene structure (vertical lines show conservation of intron position). Intron sizes are shown as number of bases and are given below vertical bars. Rab5-bind, Rabaptin 5-binding domain. TPR, tetratricopeptide repeat domain. Shaded parts indicate those genes that have been analyzed by RT-PCR and Illumina transcriptome profiling (only AaKLC2.1 is shown). Predicted introns are taken from Vectorbase.org and Ensembl.org.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927918&req=5

Figure 1: Coding sequence structure and domains of kinesin light chain genes of mosquitoes and D. melanogaster. On the left are the AaKLC2-like genes and on the right are the AaKLC1-like genes. Vertical bars show location of introns and intron size is given below bars. Note conservation of gene structure (vertical lines show conservation of intron position). Intron sizes are shown as number of bases and are given below vertical bars. Rab5-bind, Rabaptin 5-binding domain. TPR, tetratricopeptide repeat domain. Shaded parts indicate those genes that have been analyzed by RT-PCR and Illumina transcriptome profiling (only AaKLC2.1 is shown). Predicted introns are taken from Vectorbase.org and Ensembl.org.
Mentions: Structural analysis supports the categorization of two KLC gene groups, the AaKLC1-like and the AaKLC2-like genes (Figure 1). AaKLC2.1 is an intronless gene with a predicted open reading frame (ORF) of 1419 nucleotides (nt). AaKLC2.2 is also intronless and has 98% nt and aa identity to AaKLC2.1 in its coding region. The lack of introns in the AaKLC2-like genes is notable since it has been reported that 70% of Drosophila early zygotic genes do not have introns presumably for increased efficiency of transcription with fast-cycling nuclei [1]. A likely ortholog of the AaKLC2 genes was identified in C. quinquefasciatus, which is an intronless gene having a similar ORF length (gene ID: CPIJ002971). However, no similar genes were found in the genome sequences of An. gambiae [15], An. stephensi (8× coverage genome assembly, Tu unpublished), D. melanogaster, or other insects. Thus from genome analysis, it appears that AaKLC2-like genes are restricted to mosquitoes of the subfamily Culicinae. The expression of the C. quinquefasciatus gene will need to be verified by RT-PCR or other methods to determine if it is indeed an early and transient zygotic gene. This is why we have not called it CqKLC2.

Bottom Line: Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus.In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae.AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Fralin Biotech Center, Blacksburg, VA 24061, USA.

ABSTRACT

Background: The maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in Drosophila melanogaster and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as bottleneck are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito Aedes aegypti to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a Medea gene drive system.

Results: Two novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1) in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the AaKLC2.1 start codon shows promoter activity at least as early as 3 hours in the developing Ae. aegypti embryo. The AaKLC2.1 promoter activity reached ~1600 fold over the negative control at 5 hr after egg deposition.

Conclusions: Transcriptome profiling by use of high throughput sequencing technologies has proven to be a valuable method for the identification and discovery of early and transient zygotic genes. The evolutionary investigation of the KLC gene family reveals that duplication is a source for the evolution of new genes that play a role in the dynamic process of early embryonic development. AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.

Show MeSH
Related in: MedlinePlus