Non-homologous Regions Research Articles

Identifying sequence similarities between DNA molecules

An atomic force microscope (AFM) imaging technique is described to compare sequences between two different DNA molecules and precisely locate nonhomologies in DNA strands. Sequence comparisons are made by forming heteroduplexes between the two molecules and, by AFM imaging the intact molecules formed, identifying both homologous and nonhomologous regions. By forming heteroduplexes between linearized wildtype pSV- β-galactosidase plasmid (6821 bp) and a series of deletion mutants we have identified nonhomologies (deletions) as small as 22 bp and as large as 418 bp. Furthermore, by incorporating our technique for AFM-mediated restriction mapping of DNA these mutations can be positioned relative to E coRI restriction sites. These results suggest AFM can be useful in identifying molecular level similarities and differences in DNA.

A combinatorial approach to hybrid enzymes independent of DNA homology.

We present a methodology, termed incremental truncation for the creation of hybrid enzymes (ITCHY), that creates combinatorial fusion libraries between genes in a manner that is independent of DNA homology. We compared the ability of ITCHY and DNA shuffling to create interspecies fusion libraries between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, which have only 50% identity on the DNA level. Sequencing of several randomly selected positives from each library illustrated that ITCHY identified a more diverse set of active fusion points including those in regions of nonhomology and those with crossover points that diverged from the sequence alignment. Furthermore, some of the hybrids found by ITCHY that were fused at nonhomologous locations had activities that were greater than or equal to the activity of the hybrids found by DNA shuffling.

Analysis of the 5'-upstream regions of the human relaxin H1 and H2 genes and their chromosomal localization on chromosome 9p24.1 by radiation hybrid and breakpoint mapping.

Relaxins are known endocrine and autocrine/paracrine hormones that play a major role in reproduction. In the human there are two relaxin genes, H1 and H2 which share 90% sequence homology within their coding region. The biological and evolutionary significance of two highly homologous and biologically active human relaxins is unknown. In order to achieve a better understanding of the regulatory mechanisms involved in the differential expression of these two genes and to gain insight into their role(s) in the preterm premature rupture of the membranes, we have investigated the properties of their 5'-upstream regions and mapped them both by radiation hybrid and breakpoint mapping into the same chromosome 9p24.1 locus. The 5' ends of these relaxin genes could be divided into a proximal highly homologous segment and a distal non-homologous region. Within the proximal region are contained several putative regulatory elements common to both genes, suggesting a similar regulatory mechanism. The clustering of the relaxin genes within the same chromosomal locus suggests that these genes may be under a common regulation. On the other hand, a distinct gene-specific regulation may also exist for the individual relaxin genes since cis elements specific to each gene were identified at their 5' ends. Moreover, the observed divergence at the distal region of their 5'-upstream sequences may provide the structural features that act as gene-specific transcription regulators. Since the two genes are highly homologous in both their coding and flanking regions, the divergence at the distal region of their 5' ends may be important in the regulation of these genes and in their involvement in the pathology of preterm birth.

Structural requirements of Syk kinase for Fcγ receptor– phagocytosis

The tyrosine kinase Syk plays a critical role in the phagocytic pathway mediated by Fcγ receptors (FcγR). In transfected COS1 cells co-expression of Syk enhances FcγR mediated phagocytosis. The other member of the Syk kinase family, the highly homologous tyrosine kinase Zap70, also plays a role in signaling by immunoglobulin gene family receptors, but does not increase the phagocytic efficiency of FcγRs. The homologous tandem SH2 and kinase domains of Syk and Zap70 are separated by a nonhomologous region referred to as the unique domain. Zap70’s inability to enhance phagocytosis was not due to unique domain tyrosine 292, previously implicated in negative regulation of Zap70 function. We determined the regions of Syk important for its interaction with the phagocytic pathway. An intact kinase domain was required for Syk’s effect on phagocytosis. Furthermore, the Syk variant SykB, lacking 23 amino acids in the unique region, signaled for phagocytosis as efficiently as did Syk. We then constructed exchange chimeras between Syk and Zap70 and determined the contributions of the SH2, unique and kinase domains to phagocytic signaling. Our data suggest that the Syk kinase domain, which has high intrinsic kinase activity, is important for facilitating phagocytic signaling by FcγRI and FcγRIIIA.

Evidence for the presence of sex chromosomes in the Nile tilapia ( Oreochromis niloticus) from synaptonemal complex analysis of XX, XY and YY genotypes

A cytogenetic analysis of chromosome synapsis was carried out during the first meiotic prophase of the Nile tilapia, Oreochromis niloticus. Three different genotypes were studied: XX sex-reversed males, `wild-type' (XY) males and YY `supermales'. TEM analysis of synaptonemal complex (SC) spreads revealed the presence of 22 fully paired bivalents during pachytene in both homogametic genotypes. In the heterogametic genotype, an incompletely paired segment was frequently observed during the process of meiotic synapsis in the terminal region of the longest bivalent. The presence of this unpaired segment, together with several features characteristic of sex-chromosome behaviour during meiosis, suggests the existence of a non-homologous region in this chromosomal pair in the heterogametic genotype, and provides cytological evidence for the chromosomal basis of sex determination in O. niloticus. The usefulness of SC analysis for the understanding of sex determination and its relevance in the management of species of aquacultural importance are discussed.

Interactions of the α-Spectrin N-terminal Region with β-Spectrin: IMPLICATIONS FOR THE SPECTRIN TETRAMERIZATION REACTION

Spectrin of the erythrocyte membrane skeleton is composed of alpha- and beta-spectrin, which associate to form heterodimers and tetramers. It has been suggested that a fractional domain (helix C) in the amino-terminal region of alpha-spectrin (Nalpha region) bundles with another fractional domain in the carboxyl-terminal region of beta-spectrin (Cbeta region) to yield a triple alpha-helical bundle and that this helical bundling is largely responsible for tetramer formation. However, there are certain objections to assigning a preeminent role to this helical bundling in the tetramerization reactions. We prepared several recombinant peptides of alpha-spectrin fragments spanning only the Nalpha region (lacking the dimer nucleation site) and quantitatively studied their interaction with beta-spectrin. We found that a majority of the interactions were localized, as expected, in the Nalpha-helix C region but that there was also some contribution from the nonhomologous region. More importantly, the temperature and ionic strength dependence of this interaction in our model peptides was different from that in intact spectrin. We suggest that, although the regions involving the putative helical bundling in alpha- and beta-spectrin undoubtedly play a significant role in tetramerization, regions distal to the Nalpha-helix C region in spectrin are also involved in tetramer formation. Structural flexibility and lateral interactions may play a role in spectrin tetramerization.

Homology modeling of an RNP domain from a human RNA‐binding protein: Homology‐constrained energy optimization provides a criterion for distinguishing potential sequence alignments

We have recently described an automated approach for homology modeling using restrained molecular dynamics and simulated annealing procedures (Li et al, Protein Sci., 6:956–970,1997). We have employed this approach for constructing a homology model of the putative RNA-binding domain of the human RNA-binding protein with multiple splice sites (RBP-MS). The regions of RBP-MS which are homologous to the template protein snRNP U1A were constrained by “homology distance constraints,” while the conformation of the non-homologous regions were defined only by a potential energy function. A full energy function without explicit solvent was employed to ensure that the calculated structures have good conformational energies and are physically reasonable. The effects of misalignment of the unknown and the template sequences were also explored in order to determine the feasibility of this homology modeling method for distinguishing possible sequence alignments based on considerations of the resulting conformational energies of modeled structures. Differences in the alignments of the unknown and the template sequences result in significant differences in the conformational energies of the calculated homology models. These results suggest that conformational energies and residual constraint violations in these homology-constrained simulated annealing calculations can be used as criteria to distinguish between correct and incorrect sequence alignments and chain folds. Proteins 33:558–566, 1998. © 1998 Wiley-Liss, Inc.

Homology modeling of an RNP domain from a human RNA-binding protein: Homology-constrained energy optimization provides a criterion for distinguishing potential sequence alignments

We have recently described an automated approach for homology modeling using restrained molecular dynamics and simulated annealing procedures (Li et al, Protein Sci., 6:956-970,1997). We have employed this approach for constructing a homology model of the putative RNA-binding domain of the human RNA-binding protein with multiple splice sites (RBP-MS). The regions of RBP-MS which are homologous to the template protein snRNP U1A were constrained by "homology distance constraints," while the conformation of the non-homologous regions were defined only by a potential energy function. A full energy function without explicit solvent was employed to ensure that the calculated structures have good conformational energies and are physically reasonable. The effects of mis-alignment of the unknown and the template sequences were also explored in order to determine the feasibility of this homology modeling method for distinguishing possible sequence alignments based on considerations of the resulting conformational energies of modeled structures. Differences in the alignments of the unknown and the template sequences result in significant differences in the conformational energies of the calculated homology models. These results suggest that conformational energies and residual constraint violations in these homology-constrained simulated annealing calculations can be used as criteria to distinguish between correct and incorrect sequence alignments and chain folds.

Differential Regulation of Expression of Hyaluronan-Binding Proteoglycans in Developing Brain: Aggrecan, Versican, Neurocan, and Brevican

We have used a slot-blot radioimmunoassay to quantitate the levels of hyaluronan-binding chondroitin sulfate proteoglycans in developing rat brain from embryonic day 14 (E 14) to eight months postnatal. Recombinant nonhomologous regions of the core proteins were used for immunization to obtain polyclonal antibodies specific for aggrecan, the α and β domains of versican mRNA splice variants, and N- and C-terminal portions of neurocan, while brevican was quantitated using a specific monoclonal antibody. The concentration of aggrecan increased steadily during brain development up to 5 months of age, when it reached a level that was 18-fold higher than at E14. Alternatively spliced versican isoforms containing the α domain of the glycosaminoglycan attachment region were present at a relatively low level during the late embryonic and early postnatal period, decreased by ∼50% between 1 and 2 weeks postnatal, and then increased steadily in concentration to reach a maximum at 100 days that was 7-fold that present at 10 days postnatal. In contrast to these results, versican isoforms containing the β domain more than doubled in concentration between E14 and birth, after which they decreased by greater than 90% to reach a low “mature” level that remained unchanged between 2 and 8 months. The N- and C-terminal portions of neurocan (produced by a developmentally-regulated proteolytic cleavage in the middle of its chondroitin sulfate attachment region) both increased in embryonic brain during development, reached a peak in the early postnatal period, and then declined thereafter. As in the case of aggrecan, only traces of brevican were detected in embryonic brain and its concentration increased steadily after birth to reach an adult level that was approximately 14-fold higher than that present in neonatal brain. These striking and distinctive changes in the concentrations of the different members of this family of structurally related proteoglycans in developing brain, including changes in opposite directions for versican mRNA splice variants, indicate that the individual proteoglycans and their isoforms probably serve unique functions during nervous tissue histogenesis.

Wasted by an elongation factor

Some features of the wasted mouse phenotype are shared with the human autosomal recessive disorders ataxia–telangiectasia (AT) (caused by defects in the ATM gene) and Nijmegen breakage syndrome (NBS). These are immunodeficiency, ataxia, chromosomal instability and hypersensitivity of lymphocytes to ionizing radiation. However, an important difference is that fibroblasts from AT and NBS patients are hypersensitive to ionizing radiation, whereas fibroblasts derived from wasted mice exhibit normal sensitivity compared with those of wild-type mice[12xNordeen, S.K. et al. Mutat. Res. 1984; 140: 219–222Crossref | PubMed | Scopus (15)See all References][12]. This might suggest that there are more functional similarities between NBS and AT proteins than between NBS and EF1α2 proteins.Initially, the wasted mouse was suggested to be a mouse model for AT (Ref. [1xShultz, L.D., Sweet, H.O., Davisson, M.T., and Coman, D.R. Nature. 1982; 297: 402–404Crossref | PubMed | Scopus (69)See all References][1]), although AT and wasted were shown subsequently to map to non-homologous regions of the human and mouse genomes. Studies on ATM gene-targeted mice suggest that ATM is involved in the checkpoint machinery of the cell cycle, in signal transduction and in maintaining genome stability, particularly in response to DNA damage following ionizing radiation[13xLavin, M.F. and Shiloh, Y. Annu. Rev. Immunol. 1997; 15: 177–202Crossref | PubMed | Scopus (462)See all References][13]. It has been suggested that ATM could have a related function with the Saccharomyces cerevisiae signal-transduction protein Tor2p, based on its similar sequence. Inhibition of Tor2p causes an early block in translation and several other physiological changes characteristic of starved cells entering senescence[13xLavin, M.F. and Shiloh, Y. Annu. Rev. Immunol. 1997; 15: 177–202Crossref | PubMed | Scopus (462)See all References][13]. Therefore, the ATM and wst genes might be components of a signal-transduction pathway that regulates protein synthesis; the biological similarities between AT and wasted mice might partly arise from disruption of this pathway. Or, it might be possible that neurons and T cells use common apoptotic pathways and the concomitant neurodegeneration and immunodeficiency observed in AT and wst/wst is secondary to the defects in the ATM and wst genes. This common pathway could be triggered by cytokine imbalance seen in wasted mice and AT patients[14xLynn, W.S. and Wong, P.K. FASEB J. 1995; 9: 1147–1156PubMedSee all References, 15xLibertin, C.R., Ling-Indeck, L., Padilla, M., and Woloschak, G.E. Mol. Immunol. 1994; 31: 753–759Crossref | PubMed | Scopus (8)See all References].The important roles of translation elongation factors in cell cycle and cell growth have been well documented[16xRyazanov, A.G., Rudkin, B.B., and Spirin, A.S. FEBS Lett. 1991; 285: 170–175Crossref | PubMed | Scopus (67)See all References][16]. Of particular interest is the implication of these proteins in transformation and tumourigenesis[17xSonenberg, N. Curr. Opin. Cell Biol. 1993; 5: 955–960Crossref | PubMedSee all References][17]. For example, a putative oncogene, PTI1 (prostatic carcinoma tumour-inducing gene), shares strong sequence similarity to a truncated and mutated form of human Eef1a1 (Ref. [18xShen, R. et al. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6778–6782Crossref | PubMed | Scopus (103)See all References][18]). In addition, overexpression of Eef1g, the product of which, EF1γ< is a subunit of the EF1 complex, has been described in several malignancies, such as oesophageal carcinoma, adenocarcinomas and adenomas of the colon and stomach (Ref. [19xMimori, K. et al. Gut. 1996; 38: 66–70Crossref | PubMedSee all References][19]and references therein). Therefore, it would be worth investigating possible roles of other components of EF1 elongation factor complex such as EF1α, EF1β and EF1δ, in malignancies.It has been shown that in ageing Drosophila melanogaster there appears to be a reduction in the synthesis of EF1α and a subsequent decline in overall protein synthesis[20xWebster, G.C. and Webster, S.L. Mech. Ageing Dev. 1984; 24: 335–342Crossref | PubMed | Scopus (19)See all References][20]. Conversely, overexpression of EF1α seems to increase the fly life span[21xShepherd, J. et al. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7520–7521Crossref | PubMedSee all References][21]. Two genes have been described in Drosophila that encode isoforms of EF1α (Ref. [22xHovemann, B., Richter, S., Walldorf, U., and Cziepluch, C. Nucleic Acids Res. 1988; 16: 3175–3194Crossref | PubMed | Scopus (136)See all References][22]) and it would be interesting to see if the expression of either one in terminally differentiated cells protects these cells in some way and so delays or ameliorates the effects of ageing.The association of Eef1a2 with the wasted mutation is a reminder that we still have a surprising amount to learn about fundamental cellular mechanisms, such as translation, and the proteins involved in these processes. We look forward to the generation of the Eef1a2 gene-targeted mouse and further insights into the roles of translation elongation factors.

Characterization of the Enhancer-Like Okadaic Acid Response Element Region of the Cyclin-Dependent Kinase 1 (p34cdc2) Promoter

Expression of the cdk1 (p34cdc2) gene is enhanced 5–10 fold as cells re-enter the cell cycle from quiescence in response to serum-refeeding or following exposure to the protein phosphatase 1/2A inhibitor okadaic acid. Transient transfection analysis of nested deletions of the human cdk1 promoter identified regions that confer sensitivity to okadaic acid on a CAT-reporter gene. Putative okadaic acid response elements (OARE) were located between nt −942 to −763 (Site I) and nt −416 to −186 (Site II) before transcription start. The Site I element has enhancer-like characteristics as activity is independent of sequence orientation. Mobility shift analysis of Site I revealed the presence of 2 high molecular weight complexes, one of which was enhanced in the presence of okadaic acid-treated cell extracts. Site I contained several sequence motifs with conserved homology to heat shock response element core sequences and homeobox protein binding sites. Site II contained a myb-binding site, a G1/S phase enhancer, and 2 retinoblastoma response elements flanking an E2F binding site. Enhancement of cdk1 expression appears dependent on 2 nonhomologous okadaic acid-sensitive promoter regions.

Partial sequencing of Reissner's fiber glycoprotein I (RF-Gly I).

The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner's fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner's fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1. 2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner's fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner's fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.

Multiple Isoforms of Eukaryotic Protein Synthesis Initiation Factor 4E in Caenorhabditis elegans Can Distinguish between Mono- and Trimethylated mRNA Cap Structures

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5'-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m32,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegans may possess a specialized form of elF4E that can recognize m32,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, and ife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m32,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m32,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m32,2, 7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.

The involvement of cellular recombination and repair genes in RNA-mediated recombination in Saccharomyces cerevisiae.

We previously demonstrated that a reverse transcript of a cellular reporter gene (his3-AI) can serve as the donor for gene conversion of a chromosomal his3-deltaMscI target sequence, and that this process requires the yeast recombination gene RAD52. In this study, we examine the involvement of other recombination and repair genes in RNA-mediated recombination, and gain insight into the nature of the recombination intermediate. We find that mutation of the mitotic RecA homologs RAD51, RAD55, and RAD57 increases the rate of RNA-mediated recombination relative to the wild type, and that these gene functions are not required for RNA-mediated gene conversion. Interestingly, RAD1 is required for RNA-mediated gene conversion of chromosomal his3-deltaMscI sequences, suggesting that the cDNA intermediate has a region of nonhomology that must be removed during recombination with target sequences. The observation that both RAD1 and RAD52 are required for RNA-mediated gene conversion of chromosomal but not plasmid sequences indicates a clear difference between these two pathways of homologous RNA-mediated recombination.

2 α-Thalassaemia

alpha-Thalassaemias are genetic defects extremely frequent in some populations and are characterized by the decrease or complete suppression of alpha-globin polypeptide chains. The gene cluster, which codes for and controls the production of these polypeptides, maps near the telomere of the short arm of chromosome 16, within a G + C rich and early-replicating DNA region. The genes expressed during the embryonic (zeta) or fetal and adult stage (alpha 2 and alpha 1) can be modified by point mutations which affect either the processing-translation of mRNA or make the polypeptide chains extremely unstable. Much more frequent are the deletions of variable size (from approximately 3 to more than 100 kb) which remove one or both alpha genes in cis or even the whole gene cluster. Deletions of a single gene are the result of unequal pairing during meiosis, followed by reciprocal recombination. These unequal cross-overs, which produce also alpha gene triplications and quadruplications, are made possible by the high degree of homology of the two alpha genes and of their flanking sequences. Other deletions involving one or more genes are due to recombinations which have taken place within non-homologous regions (illegitimate recombinations) or in DNA segments whose homology is limited to very short sequences. Particularly interesting are the deletions which eliminate large DNA areas 5' of zeta or of both alpha genes. These deletions do not include the structural genes but, nevertheless, suppress completely their expression. Larger deletions involving the tip of the short arm of chromosome 16 by truncation, interstitial deletions or translocations result in the contiguous gene syndrome ATR-16. In this complex syndrome alpha-thalassaemia is accompanied by mental retardation and variable dismorphic features. The study of mutations of the 5' upstream flanking region has led to the discovery of a DNA sequence, localized 40 kb upstream of the zeta-globin gene, which controls the expression of the alpha genes (alpha major regulatory element or HS-40). In the acquired variant of haemoglobin H (HbH) disease found in rare individuals with myelodysplastic disorders and in the X-linked mental retardation associated with alpha-thalassaemia, a profound reduction or absence of alpha gene expression has been observed, which is not accompanied by structural alterations of the coding or controlling regions of the alpha gene complex. Most probably the acquired alpha-thalassaemia is due to the lack of soluble activators (or presence of repressors) which act in trans and affect the expression of the homologous clusters and are coded by genes not (closely) linked to the alpha genes. The ATR-X syndrome results from mutations of the XH2 gene, located on the X chromosome (Xq13.3) and coding for a transacting factor which regulates gene expression. The interaction of the different alpha-thalassaemia determinants results in three phenotypes: the alpha-thalassaemic trait, clinically silent and presenting only limited alterations of haematological parameters, HbH disease, characterized by the development of a haemolytic anaemia of variable degree, and the (lethal) Hb Bart's hydrops fetalis syndrome. The diagnosis of alpha-thalassaemia due to deletions is implemented by the electrophoretic analysis of genomic DNA digested with restriction enzymes and hybridized with specific molecular probes. Recently polymerase chain reaction (PCR) based strategies have replaced the Southern blotting methodology. The straightforward identification of point mutations is carried out by the specific amplification of the alpha 2 or alpha 1 gene by PCR followed by the localization and identification of the mutation with a variety of screening systems (denaturing gradient gel electrophoresis (DGGE), single strand conformation polymorphisms (SSCP)) and direct sequencing.

Reconstructing hominid Y evolution: X-homologous block, created by X-Y transposition, was disrupted by Yp inversion through LINE-LINE recombination.

The human X and Y chromosomes share many blocks of similar DNA sequence. We conducted mapping and nucleotide sequencing studies of extensive, multi-megabase homologies between Yp and Xq21, which do not recombine during male meiosis. We confirmed and built upon previous evidence that a Yp inversion had occurred during evolution: a single contiguous segment of Xq21 is homologous to two non-contiguous segments of Yp. We precisely defined and sequenced the inversion breakpoints, obtaining evidence that the inversion was mediated by recombination between LINE-1 elements in otherwise non-homologous regions. This inversion appears to have followed a single transposition of an approximately 4 Mb segment from the X to the Y chromosome. These events jointly account for the present arrangement of Yp-Xq21 homologous sequences. Based on Southern blotting studies of primates and of humans drawn from diverse populations, we conclude that both the X-Y transposition and the subsequent, LINE-mediated Yp inversion occurred after the divergence of hominid and chimp lineages but before the radiation of extant human populations. This evolutionary scenario is consistent with our finding of 99.3 +/- 0.2% nucleotide identity between the X and Y chromosomes within the transposed region, which suggests that the transposition occurred approximately 3-4 million years ago, near the time of emergence of Homo . Comparative sequencing of the entire human X and Y chromosomes may reveal a succession of transpositions, inversions and other rearrangements underlying the complex pattern of sequence similarities between the present-day sex chromosomes. With the possible exception of cubitus valgus, phenotypic features of Turner syndrome are absent in individuals monosomic for Yp-Xq21 homologous sequences, suggesting that most of the critical 'Turner genes' are found elsewhere on the X and Y chromosomes.

A knowledge-based three dimensional model of the Photosystem II reaction center of Chlamydomonas reinhardtii

In this Minireview, a comparison of the binding niches of the PS II cofactors from several existing models of the PS II reaction center is provided. In particular, it discusses a three dimensional model of the Photosystem II (PS II) reaction center including D1, D2 and cytochrome b559 proteins from the green alga Chlamydomonas reinhardtii that was specifically generated for this Minireview. This model is the most complete to date and includes accessory chlorophyllzs, a manganese cluster, two molecules of β-carotene and cytochrome b559, all of which are essential components of the PS II reaction center. The modeling of the D1 and D2 proteins was primarily based on homology with the L and M subunits of the anoxygenic purple bacterial photosynthetic reaction centers. The non-homologous loop regions were built using a sequence specific approach by searching for the best-matched protein segments in the Protein Data Bank, and by imposing the matching conformations on the corresponding D1 and D2 regions. Cytochrome b559 which is in close proximity to D1 and D2 was tentatively modeled in α/β conformation and docked on the QB side of the PS II reaction center according to experimental suggestions. An alternate docking on the QA side is also shown for comparison. The cofactors in the PS II reaction center were modeled either by adopting the structures from the bacterial counterparts, when available, with modifications based on existing experimental data or by de novo modeling and docking in the most probable positions in the reaction center complex. The specific features of this model are the inclusion of the tetramanganese cluster (with calcium and chloride ions) in a open, C-shaped structure modeled within the D1/D2/cytochrome b559 complex with D1-D170, D1-E189, D1-D342 and D1-A344 as putative ligands; and the modeling of two cis β-carotenes and two accessory chlorophyllzs liganded by D1-H118 and D2-H117. We also analyzed residues in the model which may be involved in the D1 and D2 inter-protein interactions, as well as residues which may be involved in putative bicarbonate and water binding and transport.

K‐tree method for high‐speed spatial normalization

The general approach to spatial normalization using a deformation field is presented. Current high degree-of-freedom deformation methods are extremely time-consuming (10–40 hr), and a k-tree method is proposed to greatly reduce this time. A general k-tree method for analysis of source and target images and synthesis of deformation fields is described. The k-tree method simplifies scale control and feature extraction and matching, making it highly efficient. A two-dimensional (2-D), or quadtree, application program was developed for preliminary testing. The k-tree method was evaluated with 2-D images to test rotating ability, nonhomologous region matching, inner and outer brain-structure independence, and feasibility with human brain images. The results of these tests indicate that a three-dimensional (3-D), or octree, method is feasible. Preliminary work with an octree application program indicates that a processing time of under 10 min for 2563 image arrays is attainable on a Sun Ultra30 workstation. Hum. Brain Mapping 6:358–363, 1998. © 1998 Wiley-Liss, Inc.

K-tree method for high-speed spatial normalization

The general approach to spatial normalization using a deformation field is presented. Current high degree-of-freedom deformation methods are extremely time-consuming (10-40 hr), and a k-tree method is proposed to greatly reduce this time. A general k-tree method for analysis of source and target images and synthesis of deformation fields is described. The k-tree method simplifies scale control and feature extraction and matching, making it highly efficient. A two-dimensional (2-D), or quadtree, application program was developed for preliminary testing. The k-tree method was evaluated with 2-D images to test rotating ability, nonhomologous region matching, inner and outer brain-structure independence, and feasibility with human brain images. The results of these tests indicate that a three-dimensional (3-D), or octree, method is feasible. Preliminary work with an octree application program indicates that a processing time of under 10 min for 256(3) image arrays is attainable on a Sun Ultra30 workstation.

The X1X2Y sex chromosome system in the fish Hoplias malabaricus. II. Meiotic analyses.

The neotropical fish Hoplias malabaricus shows diversified cytotypes and may represent a group of distinct species. One of these cytotypes is characterized by 2n = 40 and 2n = 39 chromosomes in females and males, respectively, with a multiple sex chromosome system of the X1X1X2X2/X1X2Y type. The Y, representing a large chromosome in male karyotype, is derived from a translocation event between two biarmed chromosomes: one of them similar to X1 chromosome (no. 6) and another one similar to X2 chromosome (probably no. 20). Meiotic data (standard and synaptonemal complexes analyses) show 18 bivalents and one characteristic trivalent in pachytene and metaphase I spermatocytes, as well as two kinds of metaphase II cells with 19 and 20 chromosomes. The trivalent is formed by the Y, X1 and X2 chromosomes and usually presents a complete pairing in pachytene. However, trivalents with partially or fully asynapsed segments are also observed. These segments are assumed to be non-homologous regions of the X1 and X2 chromosomes without correspondence with the Y chromosome, which can heterosynapse. This behaviour of the sex trivalent leading to a fully paired structure, taken together with the close frequencies of the two spermatocyte types at metaphase II, suggests a normal pattern for male H. malabaricus meiosis, representing a stabilized multiple sex chromosome system in this species.