Athila,a new retroelement from Arabidopsis thaliana

An analysis of Arabidopsis thaliana heterochromatic regions allowed the identification of a new family of retroelements called Athila. These 10.5 kb elements, representing ca. 0.3% of the genome, present several features of retrotransposons and retroviruses. Athila elements are flanked by 1.5 kb long terminal repeats (LTR) that are themselves bounded by 5 bp perfect inverted repeats. These LTRs start and end with the retroviral consensus 5TG...CA3 nucleotides. A putative tRNA-binding site and a polypurine tract are found adjacent to the 5 and 3 LTR respectively. The central domain is composed of two long open reading frames (ORFs) of 935 and 694 amino acids. Despite several indications of recent transposition activity, the translation of these ORFs failed to reveal significant homology with proteins associated to retrotransposition. We suggest that the Athila family could result from the transduction and dispersion of a cellular gene by a retrotransposon.     

Nucleotide sequence organization in the very small genome of a tetraodontid fish, Arothron diadematus

We have investigated the sequence organization of the very small genome (DNA content/haploid cell c = 0.4-0.5 pg) of a tetraodontid fish, Arotron diadematus, by using two main experimental approaches. The first one, renaturation kinetics, showed that slowly reassociating, intermediate, fast and foldback sequences represented 87%, 7%, 5% and 1%, respectively, of A. diadematus DNA, which is, so far, the vertebrate DNA lowest in repeated sequences. The second approach, centrifugation in Cs2SO4/BAMD density gradients [BAMD = bis(acetatomercurimethyl)dioxane], showed that A. diadematus DNA can be resolved into several components, characterized by buoyant densities of 1.700, 1.704(5), 1.708, 1.702 and 1.723 g/cm3, and representing 15%, 73%, 4%, 4% and 2.5%, respectively, of total DNA. The last component comprised a satellite DNA and ribosomal DNA. A family of interspersed repeats, possibly related to the AluI family of warm-blooded vertebrates, showed an extremely specific genomic distribution, being present in only the 1.708 g/cm3 component, which it matched in base composition.     

Target site recognition by a diversity-generating retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.     

A basal Pachyrhizodontid fish (Actinopterygii, Teleostei) from the Lower Cretaceous of the Tlayúa Quarry, Central Mexico

Michin csernai gen. et sp. nov. from the Early Cretaceous (Albian) limestones of the Tlayúa Quarry, Puebla State, Central Mexico, is assigned to the Teleost clade Pachyrhizodontoidei as it possesses the enlarged inner premaxillary tooth, which is a unique synapomorphy of this clade. Additionally, the occurrence of relatively primitive characters (e.g., united parietals, angular and articular completely fused, caudal fin with five uroneurals) suggests that Michin csernai represents the most basal representative of the pachyrhizodontids.     

Biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe

Coronaviruses (CoVs) possess large RNA genomes and exist as quasispecies, which increases the possibility of adaptive mutations and interspecies transmission. Recently, CoVs were recognized as important pathogens in captive wild ruminants. This is the first report of the isolation and detailed genetic, biologic, and antigenic characterization of a bovine-like CoV from a giraffe (Giraffa camelopardalis) in a wild-animal park in the United States. CoV particles were detected by immune electron microscopy in fecal samples from three giraffes with mild-to-severe diarrhea. From one of the three giraffe samples, a CoV (GiCoV-OH3) was isolated and successfully adapted to serial passage in human rectal tumor 18 cell cultures. Hemagglutination assays, receptor-destroying enzyme activity, hemagglutination inhibition, and fluorescence focus neutralization tests revealed close biological and antigenic relationships between the GiCoV-OH3 isolate and selected respiratory and enteric bovine CoV (BCoV) strains. When orally inoculated into a BCoV-seronegative gnotobiotic calf, GiCoV-OH3 caused severe diarrhea and virus shedding within 2 to 3 days. Sequence comparisons and phylogenetic analyses were performed to assess its genetic relatedness to other CoVs. Molecular characterization confirmed that the new isolate belongs to group 2a of the mammalian CoVs and revealed closer genetic relatedness between GiCoV-OH3 and the enteric BCoVs BCoV-ENT and BCoV-DB2, whereas BCoV-Mebus was more distantly related. Detailed sequence analysis of the GiCoV-OH3 spike gene demonstrated the presence of a deletion in the variable region of the S1 subunit (from amino acid 543 to amino acid 547), which is a region associated with pathogenicity and tissue tropism for other CoVs. The point mutations identified in the structural proteins (by comparing GiCoV-OH3, BCoV-ENT, BCoV-DB2, and BCoV-Mebus) were most conserved among GiCoV-OH3, BCoV-ENT, and BCoV-DB2, whereas most of the point mutations in the nonstructural proteins were unique to GiCoV-OH3. Our results confirm the existence of a bovine-like CoV transmissible to cattle from wild ruminants, namely, giraffes, but with certain genetic properties different from those of BCoVs.     

Molecular analysis of neurogenic placode development in a basal ray-finned fish

Neurogenic placodes are transient, thickened patches of embryonic vertebrate head ectoderm that give rise to the paired peripheral sense organs and most neurons in cranial sensory ganglia. We present the first analysis of gene expression during neurogenic placode development in a basal actinopterygian (ray-finned fish), the North American paddlefish (Polyodon spathula). Pax3 expression in the profundal placode confirms its homology with the ophthalmic trigeminal placode of amniotes. We report the conservation of expression of Pax2 and Pax8 in the otic and/or epibranchial placodes, Phox2b in epibranchial placode-derived neurons, Sox3 during epibranchial and lateral line placode development, and NeuroD in developing cranial sensory ganglia. We identify Sox3 as a novel marker for developing fields of electrosensory ampullary organs and for ampullary organs themselves. Sox3 is also the first molecular marker for actinopterygian ampullary organs. This is consistent with, though does not prove, a lateral line placode origin for actinopterygian ampullary organs.     

First records of known endoparasitic species of Pseudempleurosoma Yamaguti, 1965 (Monogenoidea: Dactylogyridae) from tetraodontid and rachycentrid fish off the northern coast of the Yucatan Peninsula, Mexico

Monogenoideans infecting the rectum of the wild checkered puffer fish, Sphoeroides testudineus (Tetraodontidae), and the pyloric ceca of the cultured cobia, Rachycentron canadum (Rachycentridae), from the northern coast of the Yucatan Peninsula, Mexico, were morphologically identified as Pseudempleurosoma carangis Yamaguti, 1965 and Pseudempleurosoma gibsoni Santos, Mour?o and Cárdenas, 2001 (Dactylogyridae), respectively. Morphometric comparison between the paratypes of P. carangis and those from S. testudineus showed that the latter differ only in the length of the body, germarium, and dorsal anchors. Similarly, a small form of P. gibsoni based on body size was detected in the present study. These metric differences may be attributable to the host effect, i.e., S. testudineus/R. canadum versus Caranx lugubris (Carangidae) (type host of P. carangis) from Hawaii and Paralonchurus brasiliensis (Sciaenidae) (type host of P. gibsoni) from Brazil, or by the degree of maturity, or both. In view of these considerations, new illustrations and several supplemental observations for P. carangis and P. gibsoni are provided. The present findings also represent new geographical records, and new sites of infection, e.g., rectum and pyloric ceca, for species of Pseudempleurosoma, and the first known endoparasitic monogenoideans infecting tetraodontid and rachycentrid fishes in Mexico.     

Selective ligand recognition by a diversity-generating retroelement variable protein

Diversity-generating retroelements (DGRs) recognize novel ligands through massive protein sequence variation, a property shared uniquely with the adaptive immune response. Little is known about how recognition is achieved by DGR variable proteins. Here, we present the structure of the Bordetella bacteriophage DGR variable protein major tropism determinant (Mtd) bound to the receptor pertactin, revealing remarkable adaptability in the static binding sites of Mtd. Despite large dissimilarities in ligand binding mode, principles underlying selective recognition were strikingly conserved between Mtd and immunoreceptors. Central to this was the differential amplification of binding strengths by avidity (i.e., multivalency), which not only relaxed the demand for optimal complementarity between Mtd and pertactin but also enhanced distinctions among binding events to provide selectivity. A quantitatively similar balance between complementarity and avidity was observed for Bordetella bacteriophage DGR as occurs in the immune system, suggesting that variable repertoires operate under a narrow set of conditions to recognize novel ligands.     

Isolation of a full-length cDNA encoding polyphenol oxidase from sugarcane, a C4 grass

Polyphenol oxidase (PPO) activity in sugarcane (a C4 grass) was highest in the growing point and declined down the stalk. Sugarcane PPO with an apparent molecular mass of 45 kDa was purified to homogeneity from immature stem tissue. Western analysis of sugarcane extracts with a polyclonal antibody raised to this protein suggested it resulted from cleavage of a 60 kDa protein during purification. The antibody was used to screen a sugarcane stem cDNA library. A full-length PPO clone (sugppol) was characterised and shown to encode a 67 kDa precursor protein comprising a plastid transit sequence of 8 kDa and a mature PPO protein of 59 kDa. High levels of expression ofsugppol were detected in the growing point of the stalk and in the immature tissue immediately below it, but no message was detected in RNA from mature stem or leaf. Comparison with other PPO sequences indicated thatsugppol was significantly different to PPO genes in C3 dicotyledonous plants.     

Identification of unique transcripts from a mouse full-length, subtracted inner ear cDNA library

A small-scale full-length library construction approach was developed to facilitate production of a mouse full-length cDNA encyclopedia representing approximately 250 enriched, normalized, and/or subtracted cDNA libraries. One library produced using this approach was a subtracted adult mouse inner ear cDNA library (sIEa). The average size of the inserts was approximately 2.5 kb, with the majority ranging from 0.5 to 7.0 kb. From this library 22,574 sequence reads were obtained from 15,958 independent clones. Sequencing and chromosomal localization established 5240 clusters, with 1302 clusters being unique and 359 representing new ESTs. Our sIEa library contributed 56.1% of the 7773 nonredundant Unigene clusters associated with the four mouse inner ear libraries in the NCBI dbEST. Based on homologous chromosomal regions between human and mouse, we identified 1018 UniGene clusters associated with the deafness locus critical regions. Of these, 59 clusters were found only in our sIEa library and represented approximately 50% of the identified critical regions.     

marY2N, a LINE-like non-long terminal repeat (non-LTR) retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake.

A LINE-like non-LTR retroelement designated marY2N was cloned from the ectomycorrhizal homobasidiomycete Tricholoma matsutake. marY2N has open reading frames that correspond to gag and pol, and a putative promoter and consensus sequences common to those of the mutators from fruit flies. While it is common to T. matsutake and Tricholoma magnivelare, marY2N does not reside in any other species of Tricholoma tested.     

The cotton centromere contains a Ty3-gypsy-like LTR retroelement

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.     

Rabaulichthys suzukii, a new anthiine fish from Japan

Rabaulichthys suzukii is described as a new species of anthiine fish, family Serranidae, from three specimens collected in 5–15 m off the Izu Peninsula, Shizuoka Prefecture, Japan in February 1999 and February 2000. It represents the third species of the genus, the most characteristic feature of which is the very high dorsal fin of the male. R. suzukii is most closely related to R. altipinnis Allen from New Britain, differing in its larger size (to 64.2 mm SL, compared to 45.5 mm for R. altipinnis), higher dorsal fin (29.3% SL, compared to 26.2% for R. altipinnis), lack of a filament on the caudal-fin lobes, 19 pectoral rays (modally 20 in R. altipinnis), 9–10 + 23 gill rakers (8–9 + 21–22 in R. altipinnis), and in color. Because the Izu Peninsula collection sites have been heavily dived for many years, the recent appearance of R. suzukii is believed to the result of transport of postlarvae from a population to the south. Received: April 20, 2000 / Revised: August 28, 2000 / Accepted: September 28, 2000