Rena Lecanidou

MITOCHONDRIAL PHYLOGEOGRAPHY OF THE LAND SNAIL ALBINARIA IN CRETE: LONG-TERM GEOLOGICAL AND SHORT-TERM VICARIANCE EFFECTS

The land snail genus Albinaria exhibits an extreme degree of morphological differentiation in Greece, especially in the island of Crete. Twenty‐six representatives of 17 nominal species and a suspected hybrid were examined by sequence analysis of a PCR‐amplified mitochondrial DNA fragment of the large rRNA subunit gene. Maximum parsimony and neighbor‐joining phylogenetic analyses demonstrate a complex pattern of speciation and differentiation and suggest that Albinaria species from Crete belong to at least three distinct monophyletic groups, which, however, are not monophyletic with reference to the genus as a whole. There is considerable variation of genetic distance within and among “species” and groups. The revealed phylogenetic relations do not correlate well with current taxonomy, but exhibit biogeographical coherence. Certain small‐ and large‐scale vicariance events can be traced, although dispersal and parapatric speciation may also be present. Our analysis suggests that there was an early and rapid differentiation of Albinaria groups across the whole of the range followed by local speciation events within confined geographical areas.

Novel Features of Metazoan mtDNA Revealed from Sequence Analysis of Three Mitochondrial DNA Segments of the Land Snail Albinaria turrita (Gastropoda: Clausiliidae)

The mitochondrial DNA (mtDNA) size of the terrestrial gastropod Albinaria turrita was determined by restriction enzyme mapping and found to be approximately 14.5 kb. Its partial gene content and organization were examined by sequencing three cloned segments representing about one-fourth of the mtDNA molecule. Complete sequences of cytochrome c oxidase subunit II (COII), and ATPase subunit 8 (ATPase8), as well as partial sequences of cytochrome c oxidase subunit I (COI), NADH dehydrogenase subunit 6 (ND6), and the large ribosomal RNA (IrRNA) genes were determined. Nine putative tRNA genes were also identified by their ability to conform to typical mitochondrial tRNA secondary structures. An 82-nt sequence resembles a noncoding region of the bivalve Mytilus edulis, even though it might contain a tenth tRNA gene with an unusual 5-nt overlap with another tRNA gene. The genetic code of Albinaria turrita appears to be the same as that of Drosophila and Mytilus edulis. The structures of COI and COII are conservative, but those of ATPase8 and ND6 are diversified. The sequenced portion of thelrRNA gene (1,079 nt) is characterized by conspicuous deletions in the 5′ and 3′ ends; this gene represents the smallest coelomate IrRNA gene so far known. Sequence comparisons of the identified genes indicate that there is greater difference between Albinaria and Mytilus than between Albinaria and Drosophila. An evolutionary analysis, based on COII sequences, suggests a possible nonmonophyletic origin of molluskan mtDNA. This is supported also by the absence of the ATPase8 gene in the mtDNA of Mytilus and nematodes, while this gene is present in the mtDNA of Albinaria and Cepaea nemoralis and in all other known coelomate metazoan mtDNAs.

A complex set of early chorion DNA sequences from Bombyx mori

A follicular cDNA library from Bombyx mori (576 clones) was screened to obtain a representative sample of chorion sequences differentially expressed during the early period of choriogenesis. Ten selected sequences were characterized by Northern analysis and by dot blots of stage-specific RNA. Five of these sequences (represented in the library by 24 clones) had the developmental specificity and transcript size expected of early chorion components; two sequences (37 clones) had the developmental specificity expected of middle and late chorion components; and three sequences (16 clones) were presumed to be non-chorion, since they are constitutive or disappear at the beginning of choriogenesis. The five recovered early chorion cDNA components have been sequenced, and define three distinct subfamilies of the chorion B multigene family, and two C type families. The complexity of the early chorion genes in B. mori is discussed.

Diversity in a chorion multigene family created by tandem duplications and a putative gene-conversion event

SummaryTwo families of high-cysteine chorion proteins inBombyx mori are encoded in 15 tandemly arranged nonidentical gene pairs. It is assumed that this locus arose by duplication with subsequent sequence divergence. We have compared DNA sequences from two such neighboring pairs of genes in an attempt to understand the manner in which diversity has been generated and/or removed. A high level of sequence identity (91%–99%) was found between the repeats throughout the transcribed and flanking regions, with two significant exceptions. First, in the DNA segment encoding a conserved region of the chorion proteins, ten substitutions were detected in a 39-base-pair region. This localized region of high variability would suggest an intergene conversion-like event. Second, a length difference of 141 base pairs was detected in a region encoding the carboxy-terminal arm of the protein. This difference can be explained by three separate reiterations of single codons (3 base pairs) separated in time by duplication or triplication events.

Developmental Control and Evolution in the Chorion Gene Families Of Insects

Publisher Summary Extensive sequence information is now available for the major chorion components in both moths and flies. These proteins have proved to be numerous but belong to a small number of families. In silk moths, such as the cultivated Bombyx mori and the wild oak silk moth Antheraea polyphemus , more than 100 major chorion components of low molecular weight can be resolved by two-dimensional gel electrophoresis. The proteins are classified according to the distinctive properties of three regions: the central domain and the flanking NH 2 - and COOH-terminal ends (left and right arms). Two fundamental groups of proteins are evident, α or β. Each group shows identities ranging from less than 50% to more than 99% and is accordingly subdivided into multiple families, subfamilies, types, and copies. The α- and β-protein groups are defined by the central domain sequences. The β proteins are highly similar in the 48-residue central domain. This high conservation most probably reflects a universal requirement for a compact secondary structure in this domain, apparently consisting of eight very short antiparallel β-sheet strands alternating with β-turns.

In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors

This is the first attempt to identify regulatory elements that are involved in early choriogenesis of the silkworm Bombyx mori. A new cis element in the promoter region of five early chorion genes was identified. The consensus sequence of this element matches the consensus of the C/EBP DNA binding site. Moreover, this sequence interacts with a 70 kD protein (pX2) present in follicular nuclear extracts and complex formation exhibits early developmental specificity. There is strong evidence that this factor belongs to the C/EBP family. Surprisingly, the same protein binds with the same developmental specificity to a similar sequence of a late chorion gene promoter, which has been previously defined as the binding site for a putative late specific factor, BCFII. The possibility that pX2 and BCFII are isoforms or modifications of the same protein factor, which is presumably able to bind to the highly similar sequence elements of both early and late genes, is discussed. A hypothesis involving protein-protein interactions between C/EBP (pX2/BCFII) and GATA during choriogenesis is presented to explain the temporal specificity of chorion genes.

Sequence analysis of a small early chorion gene subfamily interspersed within the late gene locus in Bombyx mori.

A comprehensive sequence analysis of three early chorion genes (6F6.1, 6F6.2, 6F6.3) which form a small subfamily is presented. Two main features characterize this subfamily: (1) the 6F6 gene copies are β-branch genes and, unlike typical chorion genes which are organized in divergent gene pairs, they are unpaired, and (2) they are not clustered in genetic locus Ch3 but are dispersed in Ch1-2, which is about 3 to 4 centiMorgans away and contains middle and late chorion genes. Sequence comparisons show that members of this subfamily exhibit high identity values in their major coding region (94–96%) and that similarities also extend, but to a lesser degree, into their noncoding regions. The putative 6F6 promoter regions have no significant similarities with the corresponding regions of other early β-genes but quite surprisingly share common elements with middle and late genes. The main difference among the 6F6 gene introns is the presence of inserted sequences: the insert into 6F6.2 (“IR”; 248 bp) is flanked by a 102–103-bp inverted repeat, while those into 6F6.1 (“FIB”; 184 bp) and 6F6.3 (“HOPE”; 951 bp) are carried by a partial Bm1 element. HOPE has features of a non-LTR retrotransposable element. Preliminary experiments indicate that the copy number of IR and HOPE in the Bombyx mori genome is about 5,000 and 20,000, respectively. The great similarity of 6F6 genes cannot be accounted for by selective pressure but rather appears to be the result of gene-conversion-like events, which are supposed to operate frequently in middle and late chorion genes but not in other known early β-genes. Using the relative position and orientation of the 6F6 gene copies, it is possible to propose an evolutionary scheme for the formation of chorion locus Ch1-2.

Chorion gene activation and repression is dependent on BmC/EBP expression and binding to cognate cis-elements.

From the different cis-elements clustered on silkmoth chorion gene promoters, C/EBP binding sites predominate. Their sequence composition and dispersal vary amongst promoters of diverse developmental specificity. Occupancy of these sites by BmC/EBP was examined through Southwestern and ChIP assays modified to suit ovarian follicular cells. For the genes studied, binding of BmC/EBP coincided with the respective stages of transcriptional activation. However, the factor was reloaded on promoter sequences long after individual gene repression. Furthermore, suppression of BmC/EBP transcription in developing follicles resulted in de-regulation of chorion gene expression. A biphasic function of BmC/EBP, according to which it may act as both an activator and a repressor during silkmoth choriogenesis, is considered under the light of the presented data.

Three copies of the early gene 6F6 are interspersed in and around the late chorion gene cluster of Bombyx mori

SummaryThe developmentally regulated chorion genes of the silkmoth, Bombyx mori, are clustered in two genetic loci (Chl-2 and Ch3) of chromosome 2, which are separated by approximately 4 centiMorgans. Early genes are clustered in chorion locus Ch3, whereas Ch l-2 contains all of the late genes in a continuous region of 140 kb, which is sandwiched between two regions containing most of the middle chorion genes. The late-gene area has been extensively studied and was considered to contain only late genes coding for chorion proteins of exceptionally high cysteine content organized in tightly clustered α/β gene pairs. In the present paper we report the unexpected presence of three dispersed copies (6F6.1, 6F6.2, 6F6.3) of an early β-gene, which disrupts the continuity of the late locus. Hybridization data indicate that 6F6.2 corresponds to the previously characterized m6F6 cDNA clone and that 6F6.1 and 6F6.3, but not 6F6.2, are adjacent to α-type genes. Determination of the complete sequence of 6F6.1 and of the major exon of the A-gene near it shows that these two genes have a convergent rather than a divergent direction of transcription and thus do not constitute a typical gene pair. The sequence data further suggest that 6F6.1 is transcriptionally active. The three dispersed 6F6 gene copies are localized at points of inversion of the polarity of neighboring gene pairs and their locations in this locus indicate complex gene rearrangement events.

CHD1 assumes a central role during follicle development.

During Bombyx mori follicle development, fine-tuning of chorion gene expression is under the control of bidirectional promoters. In this work, we show that the silkmoth chromo-helicase/ATPase-DNA binding protein 1 (CHD1) ortholog is responsible for repositioning of nucleosomes on chorion promoters, where the factor binds specifically. Chorion genes, occupying a single chromosomal locus, rely on an almost identical set of cis elements for their differential expression. As a direct consequence of remodeling, interaction of C/EBP and TFIID with promoter elements is facilitated and ultimately leads to initiation of transcription. Appending of methylation marks to H3K4 in a temporal-specific manner is dependent on CHD1 binding to cognate cis elements and signifies gene activation. Overall, CHD1 is a critical factor for proper development of the follicular epithelium in terms of whole-cell chromatin arrangement.