Loss of transposase-DNA interaction may underlie the divergence of mariner family transposable elements and the ability of more than one mariner to occupy the same genome

Mariners are a large family of eukaryotic DNA-mediated transposable elements that move via a cut-and-paste mechanism. Several features of the evolutionary history of mariners are unusual. First, they appear to undergo horizontal transfer commonly between species on an evolutionary timescale. They can do this because they are able to transpose using only their own self-encoded transposase and not host-specific factors. One consequence of this phenomenon is that more than one kind of mariner can be present in the same genome. We hypothesized that two mariners occupying the same genome would not interact. We tested the limits of mariner interactions using an in vitro transposition system, purified mariner transposases, and DNAse I footprinting. Only mariner elements that were very closely related to each other (ca. 84% identity) cross-mobilized, and then inefficiently. Because of the dramatic suppression of transposition between closely related elements, we propose that to isolate elements functionally, only minor changes might be necessary between elements, in both inverted terminal repeat and amino acid sequence. We further propose a mechanism to explain mariner diversification based on this phenomenon.     

A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family

Protein kinase C (PKC)-related cDNA clones encode an 84 kd protein, nPKC. nPKC contains a cysteine-rich repeat sequence homologous to that seen in conventional PKCs (alpha, beta I, beta II, and gamma), which make up a family of 77-78 kd proteins with closely related sequences. nPKC, when expressed in COS cells, confers increased high-affinity phorbol ester receptor activity to intact cells. Antibodies raised against nPKC identified a 90 kd protein in rabbit brain extract as well as in extracts from COS cells transfected with the cDNA construct. nPKC shows protein kinase activity that is regulated by phospholipid, diacylglycerol, and phorbol ester but is independent of Ca2+. The structural and enzymological characteristics of nPKC clearly distinguish it from conventional PKCs, which until now have been the only substances believed to mediate the various effects of diacylglycerol and phorbol esters. These results suggest an additional signaling pathway involving nPKC.     

HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years

Drosophila telomeres have been maintained by retrotransposition for at least 60 MY, which predates the separation of extant species of this genus. Studies of D. melanogaster, D. yakuba, and D. virilis show that, in Drosophila, telomeres are composed of two non-LTR retrotransposons, HeT-A and TART. Far from being static, HeT-A and TART evolve faster than Drosophila euchromatic genes. In spite of their high rate of sequence change, HeT-A and TART maintain their basic structures and unusual individual features. The maintenance of their separate identities suggests that HeT-A and TART cooperate either in the process of retrotransposition onto the chromosome end, or in the formation of telomere chromatin by transposed DNA copies. The telomeric retrotransposons and the Drosophila genome constitute an example of a robust symbiotic relationship between mobile elements and the genome.     

Hjc resolvase is a distantly related member of the type II restriction endonuclease family

Hjc resolvase is an archaeal enzyme involved in homologous DNA recombination at the Holliday junction intermediate. However, the structure and the catalytic mechanism of the enzyme have not yet been identified. We performed database searching using the amino acid sequence of the enzyme from Pyrococcus furiosus as a query. We detected 59 amino acid sequences showing weak but significant sequence similarity to the Hjc resolvase. The detected sequences included DpnII, HaeII and Vsr endonuclease, which belong to the type II restriction endonuclease family. In addition, a highly conserved region was identified from a multiple alignment of the detected sequences, which was similar to an active site of the type II restriction endonucleases. We substituted three conserved amino acid residues in the highly conserved region of the Hjc resolvase with Ala residues. The amino acid replacements inactivated the enzyme. The experimental study, together with the results of the database searching, suggests that the Hjc resolvase is a distantly related member of the type II restriction endonuclease family. In addition, the results of our database searches suggested that the members of the RecB domain superfamily are evolutionarily related to the type II restriction endonuclease family.     

Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule.

We report the identification and molecular characterization of Dtrk, a Drosophila gene encoding a receptor tyrosine kinase highly related to the trk family of mammalian neurotrophin receptors. The product of the Dtrk gene, gp160Dtrk, is dynamically expressed during Drosophila embryogenesis in several areas of the developing nervous system, including neurons and fasciculating axons. gp160Dtrk has structural homology with neural cell adhesion molecules of the immunoglobulin superfamily and promotes cell adhesion in a homophilic, Ca2+ independent manner. More importantly, this adhesion process specifically activates its tyrosine protein kinase activity. These findings suggest that gp160Dtrk represents a new class of neural cell adhesion molecules that may regulate neuronal recognition and axonal guidance during the development of the Drosophila nervous system.     

Regulatory elements of copia retrotransposons, controlling the level of its expression in Drosophila melanogaster testes

Expression of the lacZ reporter gene under the control of five deletion derivatives of the copia regulatory region including the 5' long terminal repeat (LTR) and the 5' untranslated region (UTR) was assayed in the testes of transgenic Drosophila melanogaster males (larvae and imago). The full-length copia regulatory region (LTR + UTR) ensured expression of the reporter gene in testes of both larvae and adult males. Deletion of UTR or 3' end of LTR increased lacZ expression in the testes, whereas deletion of the 5' end of LTR increased it. This indicated that a positive regulator of copia expression is at the 5' end of LTR and that negative regulators are at the 3' end of LTR and in UTR. The effects of the fragments of the copia regulatory region on reporter gene expression in the testes in vivo did not completely coincide with the effects observed earlier in cultured cells. We suggest that this difference is due to different regulation of expression of the fusion constructs integrated into chromatin as compared to their transient expression.     

The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet

Mitochondria are 'life-essential' organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a 'loss of function' resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.     

Isolation of distantly related members in a multigene family using the polymerase chain reaction technique.

We have designed a strategy to isolate and identify genes (cDNAs) coding for distantly-related members within a large multigene family. We have used limited protein sequence information data to delineate conserved regions where members of a supergene family are related. Comparison of the nucleotide sequences of such conserved areas defined consensus sequences that were used for the synthesis of deoxynucleotide primers. Two forward and two reverse primers were synthesized, and four separate pairs of primer combinations were used under low stringency in polymerase chain reactions (PCR) to generate amplified DNA products. The PCR products were directionally cloned into the phage vector M13mp18. Each of four libraries was screened with radiolabeled PCR product generated using a pair of primers different from those used to generate the library. Using this approach on the supergene family of ligand-gated ion channels, we were able to isolate and identify two novel subunits of neurotransmitter-operated ion channels.     

Identification of a Drosophila brain-gut peptide related to the neuropeptide Y family.

A neuropeptide F (NPF) was isolated from the fruit fly, Drosophila mellanogaster, based on a radioimmunoassay for a gut peptide from the corn earworm, Helicoverpa zea. A partial sequence was obtained from the fly peptide, and a genomic sequence coding for NPF was cloned after inverse polymerase chain reaction and shown to exist as a single genomic copy. The encoded, putative prepropeptide can be processed into an amidated NPF with 36 residues that is related to invertebrate NPF's and the neuropeptide Y family of vertebrates. In situ hybridization and immunocytochemistry showed that Drosophila NPF was expressed in the brain and midgut of fly larvae and adults.     

Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split.

In this report we describe the initial characterization of murine, human, and Drosophila hesr-1 (for hairy and enhancer of split related-1) a novel evolutionary conserved family of hairy/enhancer of split homologs. Hesr-1 cDNAs display features typical of hairy and enhancer of split-type bHLH proteins including a N-terminal bHLH domain a conserved orange domain immediately C-terminal to the bHLH region. Despite their similarity to known hairy/enhancer of split homologs, hesr-1 cDNAs are divergent members of the hairy and enhancer of split bHLH family since the degree of sequence identity within the bHLH and their nearest homologs are relatively low. Moreover, the tetrapeptide motif, WRPW, which is found in all hairy and enhancer of split family members, is not present in hesr-1. Rather, a variant of this motif, YRPW, is found. Analysis of embryonic murine hesr-1 expression by in situ hybridization reveals strong expression in the somitic mesoderm, the central nervous system, the kidney, the heart, nasal epithelium, and limbs indicating a role for hesr-1 in the development of these tissues. Like the enhancer of split cDNAs in Drosophila, we show that hesr-1 expression depends critically on signaling through the notch pathway in murine embryos, suggesting that aspects of hesr-1 regulation and function might also be evolutionary conserved.     

Chromosomal organization of a sequence related to LTR-like elements of Ty1-copia retrotransposons in Avena species.

A repetitive sequence, pAs17, was isolated from Avena strigosa (As genome) and characterized. The insert was 646 bp in length and showed 54% AT content. Databank searches revealed its high homology to the long terminal repeat (LTR) sequences of the specific family of Ty1-copia retrotransposons represented by WIS2-1A and Bare. It was also found to be 70% identical to the LTR domain of the WIS2-1A retroelement of wheat and 67% identical to the Bare-1 retroelement of barley. Southern hybridizations of pAs17 to diploid (A or C genomes), tetraploid (AC genomes), and hexaploid (ACD genomes) oat species revealed that it was absent in the C diploid species. Slot-blot analysis suggested that both diploid and tetraploid oat species contained 1.3 x 10(4) copies, indicating that they are a component of the A-genome chromosomes. The hexaploid species contained 2.4 x 10(4) copies, indicating that they are a component of both A- and D-genome chromosomes. This was confirmed by fluorescent in situ hybridization analyses using pAs17, two ribosomal sequences, and a C-genome specific sequence as probes. Further, the chromosomes involved in three C-A and three C-D intergenomic translocations in Avena murphyi (AC genomes) and Avena sativa cv. Extra Klock (ACD genomes), respectively, were identified. Based on its physical distribution and Southern hybridization patterns, a parental retrotransposon represented by pAs17 appears to have been active at least once during the evolution of the A genome in species of the Avena genus.     

Definition, expression, and characterization of a protein domain in the N-terminus of pregnancy-associated plasma protein-A distantly related to the family of laminin G-like modules

Although pregnancy-associated plasma protein-A (PAPP-A), a modulator of insulin-like growth factor (IGF) activity through its cleavage of IGF-binding protein (IGFBP)-4 and -5, has been known for more than two decades, knowledge about its domain architecture is still incomplete. Using position-specific iterative BLAST, we have identified distant relatives of the PAPP-A N-terminal sequence stretch of 250 residues. We present evidence that a protein domain with weak similarity to known laminin G-like (LG) modules is contained within this region, and we propose that PAPP-A and PAPP-A2 are new and unique members in the group of LG proteins as the pappalysins represent the first examples where LG modules are associated with proteinases. Fourteen beta-strands characteristic for the LG structure were tentatively located within the PAPP-A LG (PA-LG) module using secondary structure prediction and sequence alignment. Upon mammalian expression of PAPP-A truncation mutants, we defined domain boundaries showing that PA-LG is an autonomously folding unit, which spans the first 243 residues. We were unable to express PAPP-A variants which lack the PA-LG module, suggesting a possible role in stabilization of the proteolytic domain. To obtain larger amounts of protein for functional and structural analysis, the defined PA-LG domain was expressed in bacteria and folded in vitro. In addition, the availability of recombinant PA-LG module may potentially improve diagnostic assays based on the measurement of PAPP-A antigen, and also facilitate the study of PAPP-A in animal model systems.     

Accumulation of Spock and Worf,two novel non-LTR retrotransposons,on the neo-Y chromosome of Drosophila miranda

Transposable elements constitute a major fraction of eukaryotic genomes. Here, I characterize two novel non-LTR retrotransposons, cloned from the neo-Y chromosome of Drosophila miranda. Worf is 4.1 kb in size and shows homology to the T1-2 non-LTR transposon characterized in Anopheles. Spock is 4.9 kb in size and shows similarity to the Doc element of D. melanogaster. Southern blot analysis of both elements yielded stronger signals for male DNA. In situ hybridization to polytene chromosomes revealed that both elements are accumulating on the neo-Y chromosome of D. miranda. PCR analysis was conducted to investigate the frequency of spock and worf and of the previously identified transposons, TRIM and TRAM, at individual chromosomal sites among 12 strains of D. miranda. Contrary to the observation that element frequencies are usually kept low at individual sites in Drosophila, the four transposons investigated are fixed at their genomic locations on the neo-Y chromosome. These results support the hypothesis that transposons accumulate in nonrecombining regions and may be one cause of the heteromorphism of sex chromosomes.     

Isolation and characterization of a distantly related member of the beta-gamma crystallin super gene family from Xenopus

In an attempt to isolate gamma crystallin genes from Xenopus genomic library, we have obtained seventeen clones which strongly hybridized to the gamma 1 cDNA probe from Rana temporaria. Of these four clones are characterized. A restriction map was derived by using single and multiple digests with a variety of enzymes. Hybridization selection of lens mRNAs and in vitro translation produced a protein of 26,000 daltons. These data suggest that the above clones may contain at least part of the gamma or beta crystallin sequences.