Successive and targeted DNA integrations in the Drosophila genome by Bxb1 and phiC31 integrases

At present φC31 is the only phage integrase system available for directionally regulated site-specific DNA integration in the Drosophila genome. Here we report that mycobacteriophage Bxb1 integrase also mediates targeted DNA integration in Drosophila with high specificity and efficiency. By alternately using Bxb1 and φC31, we were able to carry out multiple rounds of successive and targeted DNA integrations in our genomic engineering founder lines for the purpose of generating complex knock-in alleles.THE serine family of phage integrases such as φC31 are highly useful due to their capability of mediating site-specific and unidirectional DNA integration in heterologous systems (Groth and Calos 2004). In the past few years, φC31-mediated site-specific DNA integration has gained wide applications in Drosophila for efficient and targeted transgenesis (Groth et al. 2004; Bateman et al. 2006; Bischof et al. 2007; Markstein et al. 2008; Ni et al. 2008). In particular, we and several other groups have developed approaches that combine φC31-mediated DNA integration with gene targeting for achieving directed and efficient modifications of endogenous genomic loci in Drosophila (Gao et al. 2008; Choi et al. 2009; Huang et al. 2009a,b; Weng et al. 2009). For example, in our genomic engineering approach, a “founder line” is first generated by homologous recombination-based gene targeting that effectively replaces the target gene with a φC31-attP (“attPC”) integration site. The target locus can then be modified into virtually any desirable knock-in alleles through φC31-mediated integration of corresponding DNA constructs into the founder line (Huang et al. 2009a,b). However, DNA integration effectively destroys the original attPC site by converting it into φC31-attR (“attRC”) and φC31-attL (“attLC”) sites (Groth and Calos 2004), preventing further DNA integrations into the target locus. Nonetheless, successive DNA integrations into a target locus can be highly desirable when making sophisticated knock-in alleles that are best done by integrating multiple constructs (Ow 2007). Although it is possible to carry out such successive DNA integrations by adding extra attPC or attBC (i.e., φC31-attB) sites on the integration construct, in practice we found that the φC31 often carried out random and promiscuous recombination among multiple attBC and/or attPC sites in Drosophila (J. Huang and Y. Hong, unpublished data), making the process highly inefficient and unreliable. Thus, an additional phage integrase is necessary for successive DNA integrations in a target locus.Mycobacteriophage Bxb1 integrase (Ghosh et al. 2003; Nkrumah et al. 2006) is a serine integrase that has been shown capable of efficient site-specific integration in heterologous systems, including malaria, plants, and mammalian cells. In addition, characterized Bxb1-attP (“attPX”) and Bxb1-attB (“attBX”) integration sites not only are distinct from attPC and attBC (Ghosh et al. 2003; Nkrumah et al. 2006), but also are small sizes of ∼50 bp, which will leave small footprints before and after integrations. To test whether Bxb1 could mediate DNA integration in Drosophila, we made a transgenic vector pAttPX that carries a 52-bp attPX (“attPX-52”) (Figure 1A) and a removable w+ marker flanked by loxP sites. Through standard P-element transposition process, we obtained five independent host lines, all coincidently carrying the attPX-52 on the third chromosome. Two of them, the attPX-52#1^[w+] and attPX-52#3^[w+] lines, were converted to w^[−] by excising the w+ marker through Cre/loxP recombination (Figure 1B) (Materials and Methods). We then made a test integration construct, pGE-attBX-GFP, which carries the 46-bp attBX site and a UAS-GFP reporter (Figure 1B), and a construct pET11Bxb1polyA for in vitro synthesis of Bxb1 mRNA (Materials and Methods). pGE-attBX-GFP/Bxb1 mRNA mixtures were prepared and injected into the homozygous attPX-52 embryos using the same protocol of φC31-mediated DNA integration (Groth et al. 2004). We obtained 16 candidate lines from attPX-52#1^[w−] and 9 candidate lines from attPX-52#3^[w−] (Open in a separate windowFigure 1 Bxb1-mediated DNA integration in Drosophila. (A) Map of pAttPX (5.984 kb). pAttPX is a P-element-based transforming vector. (B) Bxb1-mediated DNA integration. w+ marker is first excised from the attPX-52^[w+] host line by Cre-mediated recombination between two flanking loxP sites. pGE-attBX-GFP plasmid is then integrated into the attPX-52^[w−] host line via Bxb1-mediated recombination between attPX and attBX. The integration converts attPX to attRX and attLX. 3′P and 5′P, 5′ and 3′ P-element sequences; w+, hsp70::white+ marker with glass multimer reporter (GMR) enhancer (Huang et al. 2008, 2009b); PX, attPX; BX, attBX; RX, attRX; LX, attLX; Amp^R, ampicillin-resistant gene.

Table 1 

Bxb1-mediated DNA integration in attPX-52 host lines and in crb-PX genomic engineering founder lines

Genome-wide search for markers associated with bovine spongiform encephalopathy

A genome-wide search for markers associated with BSE incidence was performed by using Transmission-Disequilibrium Tests (TDTs). Significant segregation distortion, i.e., unequal transmission probabilities of alleles within a locus, was found for three marker loci on Chromosomes (Chrs) 5, 10, and 20. Although TDTs are robust to false associations owing to hidden population substructures, it cannot distinguish segregation distortion caused by a true association between a marker and bovine spongiform encephalopathy (BSE) from a population-wide distortion. An interaction test and a segregation distortion analysis in half-sib controls were used to disentangle these two alternative hypotheses. None of the markers showed any significant interaction between allele transmission rates and disease status, and only the marker on Chr 10 showed a significant segregation distortion in control individuals. Nevertheless, the control group may have been a mixture of resistant and susceptible but unchallenged individuals. When new genotypes were generated in the vicinity of these three markers, evidence for an association with BSE was confirmed for the locus on Chr 5. Subscribers can view a supplementary table for this article at url:http:// link.springer-ny/link/service/journals/OO335/contents/01/3068/paper/ index.html.     

Natural selection on polymorphic malaria antigens and the search for a vaccine

Most protein antigens identified as malaria vaccine candidates are polymorphic in natural parasite populations. Current opinion is that a vaccine must be based on conserved regions of antigens, and if naturally acquired immune responses to these regions are only partially protective in humans, then the vaccine must create what is lacking in Nature. An alternative view is that a successful vaccine might need to be based on multiple allelic forms of an antigen. David Conway here shows that, far from being too pessimistic or impractical, this view offers positive ways to identify targets of protective immunity.     

Neutralization of human papillomavirus by specific nanobodies against major capsid protein L1

The human papillomavirus (HPV) is the main cause of cervical cancer in developing countries. Rapid diagnosis and initiation of treatment of the HPV infection are critical. Various methods have been employed to reduce the immunogenicity of antibodies targeting HPV serotypes. Nanobodies are the smallest fragments of naturally occurring single-domain antibodies with their antigenbinding site compromised into a single domain. Nanobodies have remarkable properties such as high stability, solubility, and high homology to the human VH3 domain. In this study, a phagemid library was employed to enrich for nanobodies against the L1 protein of the human papilloma virus. Binding reactivity of the selected clones was evaluated using phage enzyme-linked immunosorbent assay (phage-ELISA). Finally, two nanobodies (sm5 and sm8) with the best reactivity against the Gardasil vaccine and the purified HPV-16 L1 protein were expressed and purified using a Ni(+)-NTA column. The accuracy of expression and purification of the nanobodies was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting assays. In vitro studies demonstrated that neutralization was achieved by the selected nanobodies. The ease of generation and unique features of these molecules make nanobodies promising molecules for the new generation of HPV diagnosis and therapy.     

Genome-wide search for markers associated with osteochondrosis in Hanoverian warmblood horses

A genome-wide scan was performed to detect quantitative trait loci (QTLs) for osteochondrosis (OC) and osteochondrosis dissecans (OCD) in horses. The marker set comprised 260 microsatellites. We collected data from 211 Hanoverian warmblood horses consisting of 14 paternal half-sib families. Traits used were OC (fetlock and/or hock joints affected), OCD (fetlock and/or hock joints affected), fetlock OC, fetlock OCD, hock OC, and hock OCD. The first genome scan included 172 microsatellite markers. In a second step 88 additional markers were chosen to refine putative QTLs found in the first scan. Genome-wide significant QTLs were located on equine chromosomes 2, 4, 5, and 16. QTLs for fetlock OC and hock OC partly overlapped on the same chromosomes, indicating that these traits may be genetically related. QTLs reached the chromosome-wide significance level on eight different equine chromosomes: 2, 3, 4, 5, 15, 16, 19, and 21. This whole-genome scan was a first step toward the identification of candidate genome regions harboring genes responsible for equine OC. Further investigations are necessary to refine the map positions of the QTLs already identified for OC. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome-wide search for new genes controlling plasma lipid concentrations in mice and humans

PURPOSE OF REVIEW: Quantitative trait locus analysis has been used in both humans and mice for the purpose of finding new genes regulating plasma lipid levels. We review these methods and discuss new approaches that can help find quantitative trait locus genes. RECENT FINDINGS: Many quantitative trait loci have been found that regulate plasma levels for HDL cholesterol (37 in mice and 30 in humans), LDL cholesterol (25 in mice and 20 in humans) and triglycerides (19 in mice and 30 in humans). Most of the human quantitative trait loci have concordant mouse quantitative trait loci mapping to homologous regions (93% for HDL cholesterol, 100% for LDL cholesterol and 80% for triglycerides), suggesting that many genes identified in mice may also regulate the same traits in humans. New approaches based on recently developed genomic and bioinformatic technologies and resources should greatly facilitate finding these genes. SUMMARY: New genes regulating plasma lipid levels can be found in mice and then tested in humans. Some of these genes could be potential therapeutic targets for human atherosclerosis.     

Cyanogenic Eucalyptus nobilis is polymorphic for both prunasin and specific beta-glucosidases

Cyanogenesis (i.e. the evolution of HCN from damaged plant tissue) requires the presence of two biochemical pathways, one controlling synthesis of the cyanogenic glycoside and the other controlling the production of a specific degradative beta-glucosidase. The sole cyanogenic glycoside in Eucalyptus nobilis was identified as prunasin (D-mandelonitrile beta-D-glucoside) using HPLC and GC-MS. Seedlings from three populations of E. nobilis were grown under controlled conditions and 38% were found to be acyanogenic, a proportion far greater than reported for any other cyanogenic eucalypt. A detailed study of the acyanogenic progeny from a single open-pollinated parent found that 23% lacked a cyanogenic beta-glucosidase, 32% lacked prunasin and 9% lacked both. Of the remaining seedlings initially identified as acyanogenics, 27% contained either trace amounts of beta-glucosidase or prunasin, while 9% contained trace amounts of both. Results support the hypothesis that the two components necessary for cyanogenesis are inherited independently. Trace amounts are likely to result from the presence of non-specific beta-glucosidases or the glycosylation of the cyanohydrin intermediate by non-specific UDP glycosyl transferases.     

Genome-wide analysis of genes targeted by qLTG3-1 controlling low-temperature germinability in rice

The control of seed germination under environmental conditions, where plants will be grown, is important for the adaptability of plants. Low-temperature is one of the most common environmental stress factors that affect plant growth and development and places a major limit on crop productivity in cultivated areas. Previously, qLTG3-1, a major quantitative trait locus controlling low-temperature tolerance at the germination stage in rice (called low-temperature germinability) was identified, which encodes a protein of unknown function. To identify genes targeted by qLTG3-1, a genome-wide expression profiling analysis using the 44 K Rice Oligo microarray was performed. Because the expression of qLTG3-1 was dramatically increased at 1 day after incubation, the expression profiles at this time were compared between Hayamasari, which has a loss-of-function qLTG3-1 allele, and a near isogenic line with a functional allele. A total of 4,587 genes showed significant differences between their expression levels in the two lines. Most of these genes might be involved in the process of seed germination itself, and then a focus was made on qLTG3-1 dependently induced or suppressed genes, defined as ‘qLTG3-1 dependent’ genes. Twenty-nine ‘qLTG3-1 dependent’ genes with diverse functions were categorized, implying that disruption of cellular homeostasis leads to a wide range of metabolic alterations and diverse cross-talk between various signaling pathways. In particular, genes involved in defense responses were up-regulated by qLTG3-1, indicating that qLTG3-1 expression is required for the expression of defense response genes in low-temperature germinability in rice.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs

The N¹-methyl-Adenosine (m¹A58) modification at the conserved nucleotide 58 in the TΨC loop is present in most eukaryotic tRNAs. In yeast, m¹A58 modification is essential for viability because it is required for the stability of the initiator-tRNA^Met. However, m¹A58 modification is not required for the stability of several other tRNAs in yeast. This differential m¹A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m¹A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m¹A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA^Met is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m¹A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m¹A58 hypomodified tRNAs. Most m¹A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m¹A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.     

Identification and characterization of monoclonal antibodies specific for polymorphic antigenic determinants within the V2 region of the human immunodeficiency virus type 1 envelope glycoprotein.

We have identified six monoclonal antibodies (MAbs) mapping to both linear and conformation-dependent epitopes within the V2 region of the human immunodeficiency virus type 1 clone HXB10. Three of the MAbs (12b, 66c, and 66a) were able to neutralize the molecular clones HXB10 and HXB2, with titers in the range of 9.5 to 20.0 micrograms/ml. MAbs mapping to the crown of the V2 loop (12b, 60b, and 74) bound poorly to cell surface-expressed oligomeric gp120, suggesting an explanation for the poor or negligible neutralizing activity of MAbs to this region. In contrast, MAbs 12b and 60b demonstrated good reactivity with recombinant gp120 in an enzyme-linked immunosorbent assay format, suggesting differential epitope exposure between the recombinant and native forms of gp120. Cross-competition analysis of these MAbs and additional V1V2 MAbs for gp120 binding enabled us to assign the MAbs to six groups (A to F). Selection of neutralization escape mutants with MAbs 10/76b and 11/68b, belonging to nonoverlapping competition groups, identified amino acid changes at residues 165 (I to T) and 185 (D to N), respectively. Interestingly, these escape variants remained sensitive to neutralization by the nonselecting V2 MAbs. All MAbs demonstrated good recognition of IIIB viral gp120 yet failed to neutralize nonclonal stocks of IIIB. In addition, MAbs 12b and 62c bound MN and RF viral gp120, respectively, yet failed to neutralize the respective isolates. Cloning and expression of a library of gp120 and V1V2 fragments from IIIB-, MN-, and RF-infected H9 cultures identified a number of polymorphic sites, resulting in antigenic variation and subsequent loss of V2 MAb recognition. In contrast, the V3 region from the clones of the same isolates showed no amino acid changes, suggesting that the V2 region is polymorphic in long-term-passaged laboratory isolates and may account for the reduced antibody recognition observed.     

Genome-wide search for loci controlling serum IGF binding protein levels of mice.

A segregating F(2) pedigree based on two mouse lines (DU6i and DBA/2) with extremely different growth characteristics was generated to search for loci affecting serum levels of insulin-like growth factor (IGF) binding proteins (IGFBPs) and to estimate their effects on growth and body composition. DU6i is characterized by high body mass and obesity associated with hyperinsulinemia, hyperleptinemia, and elevated serum IGF-I concentrations. Furthermore, significantly elevated serum levels of IGFBP-2, IGFBP-3, and IGFBP-4 were found in DU6i vs. DBA/2 mice. Linkage analysis identified loci with major effects on the serum level of IGFBP-3 on Chromosome 5 at 58 cM (Igfbp3q1; F = 9.9) and on Chromosome 10 at 46 cM (Igfbp3q2; F = 33.8). A locus significantly influencing serum IGFBP-2 levels in males was found on Chromosome 7. Additional linkage was detected in males and females for IGFBP-2 on Chromosomes 8, 11, 14, 17, and X, and for IGFBP-4 on Chromosome 4. Additional loci affecting IGFBPs acted in a sex-specific manner. The identified loci coincide in part with chromosomal regions controlling growth and obesity. Thus, multiple genes or pleiotropic gene effects may be assumed for these chromosomal regions. The identification of quantitative trait loci for IGFBPs as subcomponents of growth regulation and differentiation will further improve the understanding of complex trait regulation.