Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm,Bombyx mori

ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.     

A homolog of the human Hermansky–Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori

Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa ² , and oa ^v ), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.     

Molybdenum cofactor deficiency causes translucent integument,male-biased lethality,and flaccid paralysis in the silkworm Bombyx mori

Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants.     

A deleted portion of one of the two xanthine dehydrogenase genes causes translucent larval skin in the oq mutant of the silkworm (Bombyx mori)

A silkworm mutant, oq, has translucent larval skin because it is deficient in xanthine dehydrogenase (XDH) activity and is unable to synthesize uric acid, which is normally accumulated in the larval epidermis and makes the skin white and opaque. Two XDH bands were found in zymograms of the silkworm fat body: an intense band (XDHalpha) and a faint one (XDHbeta). The oq mutant lacks only XDHalpha, which seemed to be the major source of XDH activity in the fat body. An 8-bp deletion found in BmXDH1, a silkworm XDH gene, generates a premature stop codon. The resulting truncated BmXDH1 protein lacks three molybdenum cofactor-binding domains necessary for enzyme activity. BmXDH2, the other XDH gene, does not show any apparent deficiencies. BmXDH1 expressed in yeast cells yielded an activity band with the same mobility as that of XDHalpha in zymograms. BmXDH1 of the oq mutant did not yield active XDH in yeast, while the activity was restored by filling in the deleted sequence. These results showed that BmXDH1 deletion in the oq mutant is responsible for the absence of significant XDH activity, resulting in the translucent larval skin of the mutant phenotype.     

A 25 bp-long insertional mutation in the BmVarp gene causes the waxy translucent skin of the silkworm,Bombyx mori

In Bombyx mori, there are more than 35 mutant strains whose larval skin color is transparent. The waxy translucent strain ow is one of the oily mutants which lack accumulation of uric acid in the epidermis. Here we performed positional cloning of the ow gene using the Bombyx draft genome sequence. For fine structure mapping, we succeeded to narrow the ow linked region to approximately 150 kb, and identified the ow candidate gene by annotation analysis and DNA sequencing. The complete cDNA sequences of the ow gene from wild-type strains were 3501 bp-long and potentially encoded a protein of 920 amino acids. We found a 25 bp-long insertion in this gene in the ow mutant strain, resulting in a frame-shift mutation and generation of a premature stop codon. A BLAST search revealed that this protein had high homology to Varp, a recently identified protein containing a vacuolar sorting protein 9 domain and ankyrin repeats, and we termed the silkworm protein BmVarp. Varp has been shown to regulate endosome dynamics, suggesting that BmVarp may play an important role in the incorporation and/or accumulation of uric acid in the epidermis.     

A single-base deletion in an ABC transporter gene causes white eyes, white eggs, and translucent larval skin in the silkworm w-3(oe) mutant

The w-3(oe) silkworm mutant has white eyes and eggs due to the absence of ommochrome pigments in the eye pigment cells and serosa cells. The mutant is also characterized by translucent larval skin resulting from a deficiency in the transportation of uric acid, which acts as a white pigment in larval epidermal cells. A silkworm homolog of the fruitfly white gene, Bmwh3, a member of ATP-binding cassette transporter superfamily, was mapped on the w-3 locus. The w-3(oe) mutant has a single-base deletion in exon 2 and a premature stop codon at the 5' end of exon 3. These results show that w-3 is equivalent to Bmwh3 and is responsible for the transportation of ommochrome precursors and uric acid into pigment granules and urate granules, respectively.     

Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea

Background

Cabbage Fusarium wilt is a major disease worldwide that can cause severe yield loss in cabbage (Brassica olerecea). Although markers linked to the resistance gene FOC1 have been identified, no candidate gene for it has been determined so far. In this study, we report the fine mapping and analysis of a candidate gene for FOC1 using a double haploid (DH) population with 160 lines and a F₂ population of 4000 individuals derived from the same parental lines.

Results

We confirmed that the resistance to Fusarium wilt was controlled by a single dominant gene based on the resistance segregation ratio of the two populations. Using InDel primers designed from whole-genome re-sequencing data for the two parental lines (the resistant inbred-line 99–77 and the highly susceptible line 99–91) and the DH population, we mapped the resistance gene to a 382-kb genomic region on chromosome C06. Using the F₂ population, we narrowed the region to an 84-kb interval that harbored ten genes, including four probable resistance genes (R genes): Bol037156, Bol037157, Bol037158 and Bol037161 according to the gene annotations from BRAD, the genomic database for B. oleracea. After correcting the model of the these genes, we re-predicted two R genes in the target region: re-Bol037156 and re-Bol0371578. The latter was excluded after we compared the two genes’ sequences between ten resistant materials and ten susceptible materials. For re-Bol037156, we found high identity among the sequences of the resistant lines, while among the susceptible lines, there were two types of InDels (a 1-bp insertion and a 10-bp deletion), each of which caused a frameshift and terminating mutation in the cDNA sequences. Further sequence analysis of the two InDel loci from 80 lines (40 resistant and 40 susceptible) also showed that all 40 R lines had no InDel mutation while 39 out of 40 S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage.

Conclusions

This work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.     

A reexamination on the deficiency of riboflavin accumulation in Malpighian tubules in larval translucent mutants of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A variety of insects accumulate high contents of riboflavin (vitamin B₂) in their Malpighian tubules (MTs). Although this process is known to be genetically controlled, the mechanism is not known. In the 1940s and the 1950s, several studies showed that riboflavin contents were low in the MTs of some Bombyx mori (silkworm) mutants with translucent larval skin mutations (e.g., w-3, od, oa, and otm) and that genes responsible for these translucent mutations also affected riboflavin accumulation in the MTs. Since the 2000s, it has been shown that the w-3 gene encodes an ABC transporter, whereas genes responsible for od, oa, and otm mutations encode for the biogenesis of lysosome-related organelles. These findings suggest that some genes of ABC transporters and biogenesis of lysosome-related organelles may control the accumulation of riboflavin in MTs. Therefore, we reexamined the effects that translucent mutations have on the accumulation of riboflavin in MTs by using the translucent and wild-type segregants in mutant strains to measure the specific effect that each gene has on riboflavin accumulation (independent of genomic background). We used nine translucent mutations (w-3^oe, oa, od, otm, Obs, oy, or, oh, and obt) even though the genes responsible for some of these mutations (Obs, oy, or, oh, and obt) have not yet been isolated. Through observation of larval MTs and measurements of riboflavin content using high-performance liquid chromatography, we found that the oa, od, otm, and or mutations were responsible for low contents of riboflavin in MTs, whereas the Obs and oy mutations did not affect riboflavin accumulation. This indicates that the molecular mechanism for riboflavin accumulation is similar but somewhat different than the mechanism responsible for uric acid accumulation in epidermal cells. We found that the genes responsible for oa, od, and otm mutations were consistent with those already established for uric acid accumulation in larval epidermis. This suggests that these three genes control riboflavin accumulation in MTs through a mechanism similar to that of uric acid accumulation, although we do not yet know why the or mutation also controls riboflavin accumulation.     

Genetic and molecular basis of fragrance in rice

Fragrance or aroma in rice is considered as a special trait with huge economic importance that determines the premium price in global trade. With the availability of molecular maps and genome sequences, a major gene for fragrance (badh2) was identified on chromosome 8. An 8-bp deletion in the exon 7 of this gene was reported to result in truncation of betaine aldehyde dehydrogenease enzyme whose loss-of-function lead to the accumulation of a major aromatic compound, 2-acetyl 1-pyrroline (2AP) in fragrant rice. However, several studies have reported exceptions to this mutation and indicated the involvement of other genetic loci in controlling fragrance trait. These studies emphasize the need to characterize the fragrance and its underlying factors in a wide range of genetic resources available for this trait. This review summarizes the new insights gained on the genetic and molecular understanding of fragrance in rice.     

Regulation of apoptosis of rbf mutant cells during Drosophila development

Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

Xp22.3 interstitial deletion: A recognizable chromosomal abnormality encompassing VCX3A and STS genes in a patient with X-linked ichthyosis and mental retardation

X-linked ichthyosis is a genetic disorder affecting the skin and caused by a deficit in the steroid sulfatase enzyme (STS), often associated with a recurrent microdeletion at Xp22.31. Most of the STS deleted patients have X-linked ichthyosis as the only clinical feature and it is believed that patients with more complex disorders including mental retardation could be present as a result of contiguous gene deletion. In fact, VCX3A gene, a member of the VCX (variable charge, X chromosome) gene family, was previously proposed as the candidate gene for X-linked non-specific mental retardation in patients with X-linked ichthyosis.     

c.G2114A MYH9 mutation (DFNA17) causes non-syndromic autosomal dominant hearing loss in a Brazilian family

We studied a family presenting 10 individuals affected by autosomal dominant deafness in all frequencies and three individuals affected by high frequency hearing loss. Genomic scanning using the 50k Affymetrix microarray technology yielded a Lod Score of 2.1 in chromosome 14 and a Lod Score of 1.9 in chromosome 22. Mapping refinement using microsatellites placed the chromosome 14 candidate region between markers D14S288 and D14S276 (8.85 cM) and the chromosome 22 near marker D22S283. Exome sequencing identified two candidate variants to explain hearing loss in chromosome 14 [PTGDR – c.G894A:p.R298R and PTGER2 – c.T247G:p.C83G], and one in chromosome 22 [MYH9, c.G2114A:p.R705H]. Pedigree segregation analysis allowed exclusion of the PTGDR and PTGER2 variants as the cause of deafness. However, the MYH9 variant segregated with the phenotype in all affected members, except the three individuals with different phenotype. This gene has been previously described as mutated in autosomal dominant hereditary hearing loss and corresponds to DFNA17. The mutation identified in our study is the same described in the prior report. Thus, although linkage studies suggested a candidate gene in chromosome 14, we concluded that the mutation in chromosome 22 better explains the hearing loss phenotype in the Brazilian family.     

Mutations of the silkworm molybdenum cofactor sulfurase gene,og, cause translucent larval skin

Normal silkworms (Bombyx mori) have opaque larval skin due to uric acid accumulation in the epidermis while a mutant, og, is translucent owing to a deficiency in xanthine dehydrogenase (XDH), which synthesizes uric acid. Molybdenum cofactor (MoCo) sulfurase is responsible for XDH activation in various organisms. A silkworm MoCo sulfurase gene was cloned and found to be on the og locus, whose mutant alleles, og(k) and og(t), show premature stop codons, proving that og is the MoCo sulfurase gene. It was observed that a miniature inverted-repeat transposable element (MITE), named Organdy, when inserted in an og(t) mutant allele exon, causes unstable splicing of a downstream intron leading to incomplete open reading frames.     

Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm “Ming” lethal egg mutant

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive “Ming” lethal egg mutants (l-e^m) lose water and become concaved around 1 h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e^m mutant to 360 kb on chromosome 10 using 2287 F₂ individuals. Using expression analysis and RNA interference, the best l-e^m candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~ 1.9 kb region from the 3′ untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a > 100 kb DNA fragment in the l-e^m mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e^m mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e^m mutant. These results indicate that BmEP80 is responsible for the l-e^m mutation.     

Structure and specific detection of staphylococcal cassette chromosome mec type VII

Staphylococcal cassette chromosome mec (SCCmec) type VII, found in community-acquired methicillin-resistant Staphylococcus aureus belonging to multilocus sequence type (ST) 59 from Taiwan, was 41,347 bp in size and flanked by 19-bp attL and attR sequences. It was inserted into the att site at the 3′-end of orfX in the orfX-orfY (putative tRNA dihydrouridine synthase) region in ST59 S. aureus. The 5′-end side 9911-bp core region of SCCmecVII, which contained attL and the cassette chromosome recombinase gene (ccrC8), was shared by other SCC structures, SCCmercury and mosaic SCCmec from Switzerland, indicating its important role in SCC evolution. The central 21,245-bp core region contained mec complex (C2b) and another ccrC gene (ccrC2), and was highly homologous to SCCmecV, but with substitutions, insertion and replacement. The 3′-end side 10,191-bp sequence was unique. Therefore, SCCmecVII has emerged through recombination and insertion events. Multiplex and real-time PCR assays were developed for specific detection of SCCmecVII.