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We report here a case of postoperative recurrent adenosquamous carcinoma (ASC) of the esophagogastric junction (EGJ) treated with S-1 therapy. A 79-year-old woman was diagnosed with carcinoma of the EGJ. Thoracoscopic subtotal esophagectomy was performed, and pathological examination revealed advanced ASC with lymph node metastasis. Five months after surgery, multiple lung metastases and multiple lymph node metastases were observed, and the patient was treated with S-1 monotherapy, which showed partial response and may be effective for advanced ASC of the EGJ. On the other hand, immunohistological analysis of the tumors showed a relatively wide range of areas that could differentiate into both adenocarcinoma and squamous cell carcinoma, suggesting that tumor cells with multidifferentiation potential, or at least the ability to differentiate into both adeno-epithelial and squamous epithelial cells, were the likely source of the tumors., Competing Interests: None declared., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2024.)

Notwithstanding the rapid developments in sequencing techniques, Y and W sex chromosomes have still been mostly excluded from whole genome sequencing projects due to their high repetitive DNA content. Therefore, Y and W chromosomes are poorly described in most species despite their biological importance. Several methods were developed for identifying Y or W-linked sequences among unmapped scaffolds. However, it is not enough to discover functional regions from short unmapped scaffolds. Here, we provide a new and simple strategy based on k-mer comparison for comprehensive analysis of the W chromosome in Bombyx mori. Using this novel method, we effectively assembled de novo 1281 W-derived genome contigs (totaling 1.9 Mbp), and identified 156 W-linked transcript RNAs and 345 W-linked small RNAs. This method will help in the elucidation of mechanisms of sexual development and exploration of W chromosome biological functions, and provide insights into the evolution of sex chromosomes. Moreover, we showed this method can be employed in identifying heterogametic sex chromosomes (W and Y chromosomes) in many other species where genomic information is still scarce.

Because self-incompatibility loci are maintained heterozygous and recombination within self-incompatibility loci would be disadvantageous, self-incompatibility loci are thought to contribute to structural and functional differentiation of chromosomes. Although the hermaphrodite chordate, Ciona intestinalis, has two self-incompatibility genes, this incompatibility system is incomplete and self-fertilization occurs under laboratory conditions. Here, we established an inbred strain of C. intestinalis by repeated self-fertilization. Decoding genome sequences of sibling animals of this strain identified a 2.4-Mbheterozygous region on chromosome 7. A self-incompatibility gene, Themis-B, was encoded within this region. This observation implied that this self-incompatibility locus and the linkage disequilibrium of its flanking region contribute to the formation of the 2.4-Mb heterozygous region, probably through recombination suppression. We showed that different individuals in natural populations had different numbers and different combinations of Themis-B variants, and that the rate of self-fertilization varied among these animals. Our result explains why self-fertilization occurs under laboratory conditions. It also supports the concept that the Themis-B locus is preferentially retained heterozygous in the inbred line and contributes to the formation of the 2.4-Mb heterozygous region. High structural variations might suppress recombination, and this long heterozygous region might represent a preliminary stage of structural differentiation of chromosomes., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

The SilkDB is an open-access database for genome biology of the silkworm (Bombyx mori). Since the draft sequence was completed and the SilkDB was first released 5 years ago, we have collaborated with other groups to make much remarkable progress on silkworm genome research, such as the completion of a new high-quality assembly of the silkworm genome sequence as well as the construction of a genome-wide microarray to survey gene expression profiles. To accommodate these new genomic data and house more comprehensive genomic information, we have reconstructed SilkDB database with new web interfaces. In the new version (v2.0) of SilkDB, we updated the genomic data, including genome assembly, gene annotation, chromosomal mapping, orthologous relationship and experiment data, such as microarray expression data, Expressed Sequence Tags (ESTs) and corresponding references. Several new tools, including SilkMap, Silkworm Chromosome Browser (SCB) and BmArray, are developed to access silkworm genomic data conveniently. SilkDB is publicly available at the new URL of http://www.silkdb.org.

Juvenile hormone epoxide hydrolases (JHEHs) are a family of enzymes that hydrolyze juvenile hormones (JHs). They are important in terms of organ-specific regulation and irreversible degradation. In contrast to three JHEH genes (jheh) in Drosophila melanogaster and five jheh in Tribolium castaneum, only one jheh gene has been reported to date in lepidopteran insects. By searching a genome database of the silkworm, KAIKOBLAST, five JHEH-related genes (jheh-r), in addition to Bmjheh, were found. Developmental changes in mRNA expression were brought about revealing several unique patterns for each of jheh-r as to developmental stages and organ-specificity. Recombinant proteins of JHEH-r were expressed using a baculovirus system to evaluate their enzymatic activities. Three of the five JHEH-r recombinant proteins had JH hydrolytic activities. This is the first report on lepidopteran jheh-related genes and also provides the comprehensive analysis of multiple jheh-related genes in an insect species with respect to their functions in enzyme activities.

The role of sex chromosomes in sex determination has been well studied in diverse groups of organisms. However, the role of the genes on the sex chromosomes in conferring sexual dimorphism is still being experimentally evaluated. An unequal complement of sex chromosomes between two sexes makes them amenable to sex-specific evolutionary forces. Sex-linked genes preferentially expressed in one sex over the other offer a potential means of addressing the role of sex chromosomes in sexual dimorphism. We examined the testis transcriptome of the silkworm, Bombyx mori, which has a ZW chromosome constitution in the female and ZZ in the male, and show that the Z chromosome harbors a significantly higher number of genes expressed preferentially in testis compared to the autosomes. We hypothesize that sexual antagonism and absence of dosage compensation have possibly led to the accumulation of many male-specific genes on the Z chromosome. Further, our analysis of testis-specific paralogous genes suggests that the accumulation on the Z chromosome of genes advantageous to males has occurred primarily by translocation or tandem duplication.

We describe our findings on HLH54F, a basic helix-loop-helix transcription factor gene that was highly expressed in the prothoracic gland, an organ producing the insect steroid ecdysone. HLH54F was uncovered by the use of an expressed sequence tag database of the silkworm Bombyx mori. It was also highly expressed in the prothoracic gland of the fruit fly Drosophila melanogaster.

Many larval color mutants have been obtained in the silkworm Bombyx mori. Mapping of melanin-synthesis genes on the Bombyx linkage map revealed that yellow and ebony genes were located near the chocolate (ch) and sooty (so) loci, respectively. In the ch mutants, body color of neonate larvae and the body markings of elder instar larvae are reddish brown instead of normal black. Mutations at the so locus produce smoky larvae and black pupae. F(2) linkage analyses showed that sequence polymorphisms of yellow and ebony genes perfectly cosegregated with the ch and so mutant phenotypes, respectively. Both yellow and ebony were expressed in the epidermis during the molting period when cuticular pigmentation occurred. The spatial expression pattern of yellow transcripts coincided with the larval black markings. In the ch mutants, nonsense mutations of the yellow gene were detected, whereas large deletions of the ebony ORF were detected in the so mutants. These results indicate that yellow and ebony are the responsible genes for the ch and so loci, respectively. Our findings suggest that Yellow promotes melanization, whereas Ebony inhibits melanization in Lepidoptera and that melanin-synthesis enzymes play a critical role in the lepidopteran larval color pattern.

Introduction: Some Japanese women are known to have negative attitudes toward erectile dysfunction (ED) drugs, but the environment underlying these perceptions is unclear., Aim: To reveal the underlying environment that shapes women's perceptions of ED drugs in Japan., Methods: A web-based questionnaire was conducted through an Internet-based market research company. A total of 2,593 women in five age groups (20s, 30s, 40s, 50s, and 60s or older) were randomly invited to participate in this study, with an almost equal number in each age group. The questionnaire contained 30 items related to individual background and sexual information, concerns about the image of ED drugs, ED drug-related perceptions, and attitude toward sexual information media., Main Outcome Measures: The women's attitude and the independent predictors that affect their partner's use of ED drugs were clarified., Results: Answers were obtained from 1,077 women, of whom 35.4% (n=381) had a negative image of ED drugs. Although 69.5% (n=749) agreed that a sexual relationship with a male partner was important, only 26.7% (n=288) agreed that this remained important if ED drugs were used. However, 56.7% (n=611) and 57.7% (n=621) of respondents, respectively, answered that they would allow their partner's use of ED drugs if they imagined that they understood the safety and effectiveness of ED drugs and that their quality of life was improved by their partner's use of the drugs. Lack of information about ED drugs was a significant predictor for acceptance of a partner's use of ED drugs among women with a negative image of ED drugs, since they were more likely to accept the use of these drugs if they were convinced about their safety and effectiveness or positive effect on quality of life., Conclusion: Lack of information about ED drugs may influence the perception of women in Japan regarding these drugs.

Mutations at the flügellos (fl) locus in Bombyx mori produce wingless pupae and moths because of the repressed response of wing discs to ecdysteroid. Four recessive fl alleles occurred spontaneously and were mapped at 13.0 of the silkworm genetic linkage group 10. By positional cloning, we confirmed that the gene responsible for fl is fringe (fng) encoding Fng glycosyltransferase, which is involved in regulating the Notch signaling pathway. In four different fl alleles, we detected a large deletion of the fng gene in fl(k) and nonsense mutations in fl, fl(o), and fl(n). In the wild-type (WT) silkworm, fng is expressed actively in the wing discs, brain, and reproductive organs from the fourth to final instars but barely in the other tissues tested. In situ hybridization showed that fng mRNA is expressed in the dorsal layer of the WT wing discs. The wingless (wg) mRNA, a downstream marker of Fng-mediated Notch signaling, is localized at the dorsoventral boundary in the WT wing discs but repressed markedly in the fl wing discs. Although null mutants of Drosophila fng result in postembryonic lethality, loss of fng function in Bombyx affects only wing morphogenesis, suggesting different essential roles for fng in tissue differentiation among insects.

Hornet silk, a fibrous protein in the cocoon produced by the larva of the vespa, is composed of four major proteins. In this study, we constructed silk-gland cDNA libraries from larvae of the hornet Vespa simillima xanthoptera Cameron and deduced the full amino acid sequences of the four hornet silk proteins, which were named Vssilk 1-4 in increasing order of molecular size. Portions of the amino acid sequences of the four proteins were confirmed by Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and N-terminal protein sequencing. The primary sequences of the four Vssilk proteins (1-4) were highly divergent, but the four proteins had some common properties: (i) the amino acid compositions of all four proteins were similar to each other in that the well-defined and characteristic repetitive patterns present in most of the known silk proteins were absent; and (ii) the characteristics of the amino acid sequences of the four proteins were also similar in that Ser-rich structures such as sericin were localized at both ends of the chains and Ala-rich structures such as fibroin were found in the center. These characteristic primary structures might be responsible for the coexisting alpha-helix and beta-sheet conformations that make up the unique secondary structure of hornet silk proteins in the native state. Because heptad repeat sequences of hydrophobic residue are present in the Ala-rich region, we believe that the Ala-rich region of hornet silk predominantly forms a coiled coil with an alpha-helix conformation.

Background: Expression of estrogen-regulated genes has been considered as potential predictive markers for endocrine therapy. We focused on two insulin-like growth factor binding proteins (IGFBPs): IGFBP-4, which is an early-responsive estrogen-induced gene, and IGFBP-5, which is an estrogen-repressed gene. Investigation of IGFBP-4 and IGFBP-5 expression would provide important information for predicting prognosis and endocrine responsiveness., Methods: The levels of IGFBP-4 and IGFBP-5 mRNA expression in 162 human breast cancer tissues were analyzed using quantitative real-time reverse transcriptase-PCR. The association between IGFBP-4 and IGFBP-5 expression and clinicopathological factors was then analyzed., Results: The levels of IGFBP-4 and IGFBP-5 mRNA expression were positively correlated with estrogen receptor (ER) and progesterone receptor (PgR) status and were negatively correlated with HER2 overexpression. Patients with a high level of IGFBP-4 mRNA expression had better disease-free and overall survival than those with a low expression. Multivariate analysis showed that IGFBP-4 mRNA expression is an independent prognostic factor for disease-free survival. When analyzed in 116 patients with ER-positive breast cancer, patients whose tumor expressed higher levels of IGFBP-4 mRNA or lower levels of IGFBP-5 mRNA had better disease-free survival., Conclusion: IGFBP-4 mRNA expression was an independent prognostic factor in breast cancer, and patients with ER-positive breast cancer whose tumor expressed higher levels of IGFBP-4 and lower levels of IGFBP-5 had a better prognosis than those without such findings.

Objective: To clarify the impact of radical perineal prostatectomy (RPP) on lower urinary tract symptoms (LUTS) in patients with clinically localized prostate cancer., Methods: A total of 117 patients with a median age of 69 years who underwent RPP alone between August 2002 and August 2005 were included in the study. We measured LUTS on the basis of the International Prostate Symptom Score (IPSS) and IPSS quality of life (QOL) questionnaire before, and 3, 6 and 12 months after surgery., Results: The overall mean total IPSS and IPSS QOL score decreased over time after RPP and was significantly reduced at 12 months after surgery. The decrease of the score was more prominent and rapid in patients with moderate to severe symptoms (IPSS > or = 8), whereas in those with no or only mild symptoms (IPSS < or = 7), the score did not change significantly after RPP. When the patients were divided into groups with baseline scores of 0-1 and 2-5 for each of the seven composites of the IPSS, scores for the 2-5 group improved significantly after RPP in all composites, whereas the 0-1 group had significantly worse scores for voiding frequency and nocturia. No significant change was noted in any of the other five composites., Conclusion: This longitudinal study shows that RPP is significantly beneficial for moderate to severe LUTS, but also has adverse effects on voiding frequency and nocturia in some men with no or mild symptoms. This information is important when counseling patients about treatment options for localized prostate cancer.

Beta-N-acetylglucosaminidase is a major glycosidase involved in several physiological processes, such as fertilization, metamorphosis, glycoconjugate degradation, and glycoprotein biosynthesis in insects. A search using the Bombyx mori cDNA database revealed the existence of two putative beta-N-acetylglucosaminidase genes. Their full-length cDNAs were cloned by rapid amplification of cDNA ends and polymerase chain reaction using specific primers, and named BmGlcNAcase1 and BmGlcNAcase2. A BLAST search revealed that BmGlcNAcase1 and BmGlcNAcase2 are homologous to a beta-subunit homolog encoded by Drosophila melanogaster HEXO2 and the Spodoptera frugiperda beta-N-acetylglucosaminidase gene respectively. The recombinant proteins of BmGlcNAcase1 and BmGlcNAcase2 without putative transmembrane domains were expressed in the yeast Pichia pastoris. Both enzymes showed broad substrate specificity, and cleaved terminal N-acetylglucosamine residues from the alpha-3 and alpha-6 branches of a biantennary N-glycan substrate, and also hydrolyzed chitotriose to chitobiose.

Objective: The aim of this study was to clarify whether intravesical recurrence of upper urinary tract cancer after treatment is related to the mode of surgery or other oncological factors., Methods: We evaluated 106 patients (mean age 70.4 years; mean follow-up 24.0 months) who underwent surgery for the upper urinary tract cancer at Hiroshima University and its affiliated hospitals between January 1995 and August 2005. Seventy-nine of the patients underwent retroperitoneoscopy-assisted radical nephroureterectomy (RN) and 27 underwent nephroureterectomy by open surgery (OS). Fifty-two patients had renal pelvic tumors, 48 had ureteral tumors, and six had both renal pelvic and ureteral tumors. Twenty-eight (26%) of the 106 patients had a pre-operative history of bladder cancer. We identified the risk factors predicting intravesical recurrence of upper urinary tract cancer according to the type of previous surgery using the Kaplan-Meier method, log-rank test, and univariate and multivariate analysis using the Cox proportional hazards model., Results: Thirty-one (29%) of the 106 patients developed bladder tumors post-operatively. The 2-year intravesical recurrence-free rate was 55% in the RN group and 60% in the OS group. There was no significant difference (P = 0.51, log-rank test) in the rate of intravesical recurrence between the two groups. Multivariate analysis identified only a history of pre-operative bladder tumor (HR = 3.25, P = 0.003) as a predictor of post-operative intravesical recurrence., Conclusions: Intravesical recurrence after surgery for upper urinary tract cancer is not related to the mode of surgery (i.e. laparoscopy-assisted or open surgery) employed. The only risk factor for intravesical recurrence is a history of bladder cancer.

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female x an F1 (p50T x C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.

The SilkSatDb (silkmoth microsatellite database) (http://www.cdfd.org.in/silksatdb) is a relational database of microsatellites extracted from the available expressed sequence tags and whole genome shotgun sequences of the silkmoth, Bombyx mori. The database has been rendered with a simple and robust web-based search facility, developed using PHP. The SilkSatDb also stores information on primers developed and validated in the laboratory. Users can retrieve information on the microsatellite and the protocols used, along with informative figures and polymorphism status of those microsatellites. In addition, the interface is coupled with Autoprimer, a primer-designing program, using which users can design primers for the loci of interest.

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of >/=15 bases of mononucleotide repeats and >/=5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2-14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.

Motivation: A whole set of Expressed Sequence Tags (ESTs) from the Sf9 cell line of Spodoptera frugiperda is presented here for the first time. By this way we want to identify both conserved and specific genes of this pest species. We also expect from this analysis to find a class of protein sequences providing a tool to explore genomic features and phylogeny of Lepidoptera., Results: The ESTs display both housekeeping as well as developmentally regulated genes, and a high percentage of sequences with unknown function. Among the identified ORFs, almost all ribosomal proteins (RPs) were found with high EST redundancy and hence sequence accuracy. The codon usage found among RP genes is in average surprisingly much less biased in Lepidoptera than in other organisms. Other Spodoptera genes also displayed a low bias, suggesting a general genome expression feature in this Lepidoptera. We also found that the L35A and L36 RP sequences, respectively, display 40 and 10 amino-acid insertions, both being present only in insects. Sequence analysis suggests that they are probably not subjected to a strong selective pressure and may be good phylogenetic markers for Lepidoptera. Most interestingly, the Lepidoptera sequences of 9 RP genes displayed a specific signature different from the canonical one. We conclude that the RP family allows valuable comparative genomics and phylogeny of Lepidoptera., Availability: All EST sequence data are available from the private 'Spodo-Base' upon request.

The polyubiquitin gene is an evolutionarily conserved eukaryotic gene, encoding tandemly repeated multiple ubiquitins, and is considered to be subject to concerted evolution. Here, we present the nucleotide sequences of new alleles of the polyubiquitin gene UbC in humans and CHUB2 in Chinese hamster, which encode a different number of ubiquitin units from those of previously reported genes. And we analyze the concerted evolution of these genes on the basis of their orthologous relationship. That the mean of the synonymous sequence difference Ks which is defined as the number of synonymous substitution relative to the total number of synonymous sites, within the UbC and CHUB2 genes (0.192 +/- 0.096) is significantly less than Ks between these genes (0.602 +/- 0.057) provides direct evidence for concerted evolution. Moreover, it also appears that concerted evolutionary events have been much more frequent in CHUB2 than in UbC, because Ks within CHUB2 (0.022 +/- 0.018) is much less than that within UbC (0.362 +/- 0.192). By a numerical simulation, postulating that the major mechanism of concerted evolution in polyubiquitin genes is unequal crossing over, we estimated the frequency of concerted evolutionary events of CHUB2 at 3.3 x 10(-5) per year and that of UbC at no more than 5.0 x 10(-7) per year.

A subtraction procedure was developed for identification and isolation of a human gene transcribed in mouse transformant cells. The procedure was based on subtractive enrichment of the products that were amplified by the combination of reverse transcription and polymerase chain reaction from the 3'-untranslated region (3'-UTR) of human poly(A)+ RNA expressed in the mouse transformant cells. To assess the ability and usefulness of the procedure, we attempted to recover the human purH gene from a mouse transformant cell line, which was originally established by functional complementation using the human metaphase chromosome-mediated gene transfer technique from a mouse purH-negative mutant cell line. Using our procedure, a part of the human transcript in the transformant cells was successfully identified and isolated. The full-length cDNA was isolated using the 3'-UTR clone as a probe, and its biological activity was confirmed by introducing it into the mouse purH-negative mutant cells.

Nucleosome core particles from chicken erythrocytes have been studied by small angle neutron scattering over the range from 10 to 0.04 mM Na+ at 65 and 100% D2O, and the radii of gyration of the particle were determined. A single transition in the radius of gyration was observed at either D2O concentration. With decreasing the ionic strength from 10 mM, the radius of gyration of the histones obtained at 65% D2O increased from 35 to 40A at about 1 mM ionic strength, whereas at 100% D2O the radius of gyration decreased from 39 to 36A also near 1 mM ionic strength. No loss of the secondary structure of the histones was observed by circular dichroism over the range of the ionic strength examined. These results suggest that at low ionic strength (less than or equal to 1 mM) the histones may locate outside of the nucleosome core particle accompanied by an alteration of the tertiary and/or the quaternary structure of the histone octamer.