Hybridization Protection Reaction for Sensitive and Robust Gene Expression Profiling of Clinical Formalin-Fixed Paraffin-Embedded Samples.

BACKGROUND: RNA profiling of formalin-fixed paraffin-embedded (FFPE) tumor tissues for the molecular diagnostics of disease prognosis or treatment response is often irreproducible and limited to a handful of biomarkers. This has led to an unmet need for robust multiplexed assays that can profile several RNA biomarkers of interest using a limited amount of specimen. Here, we describe hybridization protection reaction (HPR), which is a novel RNA profiling approach with high reproducibility. METHODS: HPR assays were designed for multiple genes, including 10 radiosensitivity-associated genes, and compared with TaqMan assays. Performance was tested with synthetic RNA fragments, and the ability to analyze RNA was investigated in FPPE samples from 20 normal lung tissues, 40 lung cancer, and 30 esophageal cancer biopsies. RESULTS: Experiments performed on 3 synthetic RNA fragments demonstrated a linear dynamic range of over 1000-fold with a replicate correlation coefficient of 0.99 and high analytical sensitivity between 3.2 to 10 000 pM. Comparison of HPR with standard quantitative reverse transcription polymerase chain reaction on FFPE specimens shows nonsignificant differences with > 99% confidence interval between 2 assays in transcript profiling of 91.7% of test transcripts. In addition, HPR was effectively applied to quantify transcript levels of 10 radiosensitivity-associated genes. CONCLUSIONS: Overall, HPR is an alternative approach for RNA profiling with high sensitivity, reproducibility, robustness, and capability for molecular diagnostics in FFPE tumor biopsy specimens of lung and esophageal cancer.

Molecular and Genetics-Based Systems for Tracing the Evolution and Exploring the Mechanisms of Human Norovirus Infections.

Human noroviruses (HuNoV) are major causes of acute gastroenteritis around the world. The high mutation rate and recombination potential of noroviruses are significant challenges in studying the genetic diversity and evolution pattern of novel strains. In this review, we describe recent advances in the development of technologies for not only the detection but also the analysis of complete genome sequences of noroviruses and the future prospects of detection methods for tracing the evolution and genetic diversity of human noroviruses. The mechanisms of HuNoV infection and the development of antiviral drugs have been hampered by failure to develop the infectious virus in a cell model. However, recent studies have demonstrated the potential of reverse genetics for the recovery and generation of infectious viral particles, suggesting the utility of this genetics-based system as an alternative for studying the mechanisms of viral infection, such as cell entry and replication.

Detection and discrimination of maintenance and de novo CpG methylation events using MethylBreak.

Understanding the principles governing the establishment and maintenance activities of DNA methyltransferases (DNMTs) can help in the development of predictive biomarkers associated with genetic disorders and diseases. A detection system was developed that distinguishes and quantifies methylation events using methylation-sensitive endonucleases and molecular beacon technology. MethylBreak (MB) is a 22-mer oligonucleotide with one hemimethylated and two unmethylated CpG sites, which are also recognition sites for Sau96I and SacII, and is attached to a fluorophore and a quencher. Maintenance methylation was quantified by fluorescence emission due to the digestion of SacII when the hemimethylated CpG site is methylated, which inhibits Sau96I cleavage. The signal difference between SacII digestion of both MB substrate and maintenance methylated MB corresponds to de novo methylation event. Our technology successfully discriminated and measured both methylation activities at different concentrations of MB and achieved a high correlation coefficient of R2=0.997. Additionally, MB was effectively applied to normal and cancer cell lines and in the analysis of enzymatic kinetics and RNA inhibition of recombinant human DNMT1.

Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response.

BacMam is an insect-derived recombinant baculovirus that can deliver genes into mammalian cells. BacMam vectors carrying target genes are able to enter a variety of cell lines by endocytosis, but the level of expression of the transgene depends on the cell line and the state of the transduced cells. In this study, we demonstrated that the DNA damage response (DDR) could act as an alternative pathway to boost the transgene(s) expression by BacMam and be comparable to the inhibitors of histone deacetylase. Topoisomerase II (Top II) inhibitor-induced DDR can enhance the CMV-IE/enhancer mediated gene expression up to 12-fold in BacMam-transduced U-2OS cells. The combination of a Top II inhibitor, VM-26, can also augment the killing efficiency of a p53-expressing BacMam vector in U-2OS osteosarcoma cells. These results open a new avenue to facilitate the application of BacMam for gene delivery and therapy.

The effect of chemically modified electrospun silica nanofiber on the mRNA and miRNA expression profile of neural stem cell differentiation.

A detailed genomic and epigenomic analyses of neural stem cells (NSCs) differentiation in synthetic microenvironments is essential for the advancement of regenerative medicine and therapeutic treatment of diseases. This study identified the changes in mRNA and miRNA expression profile during NSC differentiation on an artificial matrix. NSCs were grown on a surface-modified, electrospun tetraethyl-orthosilicate nanofiber (designated as SNF-AP) by providing a 3D-environment for cell growth and differentiation. Differentially expressed mRNAs and miRNAs of NSC differentiated in this microenvironment were identified through microarray analysis. The genes and miRNA targets responsible for the differentiation fate of NSCs and neuron development process were determined using Ingenuity Pathway Analysis (IPA). SNF-AP enhanced the expression of genes that activates the proliferation, development, and outgrowth of neurons, differentiation and generation of cells, neuritogenesis, outgrowth of neurites, microtubule dynamics, formation of cellular protrusions, and long-term potentiation during NSC differentiation. On the other hand, PDL inhibited neuritogenesis, microtubule dynamics, and proliferation and differentiation of cells and activated the apoptosis function. Moreover, the nanomaterial promoted the expression of more let-7 miRNAs, which have vital roles in NSC differentiation. Overall, SNF-AP is biocompatible and applicable scaffold for NSC differentiation in the development of neural tissue engineering. These findings are useful in enhancing in vitro NSC differentiation potential for preclinical studies and future clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2730-2743, 2016.

Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein.

UNLABELLED: Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3' untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication. IMPORTANCE: Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.

A novel sensitive pathogen detection system based on Microbead Quantum Dot System.

A fast and accurate detection system for pathogens can provide immediate measurements for the identification of infectious agents. Therefore, the Microbead Quantum-dots Detection System (MQDS) was developed to identify and measure target DNAs of pathogenic microorganisms and eliminated the need of PCR amplifications. This nanomaterial-based technique can detect different microorganisms by flow cytometry measurements. In MQDS, pathogen specific DNA probes were designed to form a hairpin structure and conjugated on microbeads. In the presence of the complementary target DNA sequence, the probes will compete for binding with the reporter probes but will not interfere with the binding between the probe and internal control DNA. To monitor the binding process by flow cytometry, both the reporter probes and internal control probes were conjugated with Quantum dots that fluoresce at different emission wavelengths using the click reaction. When MQDS was used to detect the pathogens in environmental samples, a high correlation coefficient (R=0.994) for Legionella spp., with a detection limit of 0.1 ng of the extracted DNAs and 10 CFU/test, can be achieved. Thus, this newly developed technique can also be applied to detect other pathogens, particularly viruses and other genetic diseases.

Genetic Diagnosis via Whole Exome Sequencing in Taiwanese Patients with Hypertriglyceridemia.

AIM: Whole exome sequencing (WES) is a recently developed method for discovering rare mutations associated with hereditary disorders. However, the feasibility and utilization of this method in identifying familial hypertriglyceridemia is not well known. The purpose of the study was to identify the genetic locus that causes hypertriglyceridemia and assess its prevalence in Taiwanese subjects with hypertriglyceridemia. METHODS: We performed WES among two individuals with hypertriglyceridemia and one control subject in an index family (22 members). Based on the WES findings, we extended the study to genotype 65 unrelated adult index patients with a fasting serum triglyceride level of ＞ 500 mg/dL and 125 normal controls using polymerase chain reaction. RESULTS: Using WES alignment, variant calling and annotation, 15 presumptive causal variants were initially identified, including 13 cases by the autosomal dominant model and two cases by the autosomal recessive model. Only APOA5 c.553 G ＞ T (rs2075291), resulting in the amino acid mutation Gly185Cys, co-segregated well with hypertriglyceridemia in terms of autosomal recessive inheritance (homozygote TT: mean triglyceride level: 1,071 mg/dL vs non TT (GT and GG): mean triglyceride level: 118 mg/dL; p ＜ 0.001) in the index family. In the unrelated cohorts, the frequency of the TT genotype of rs2075291 was 12.3% in the hypertriglyceridemic group; however, no TT genotype was found in the control group. CONCLUSIONS: Our results demonstrate that WES is feasible for identifying the genetic locus that causes hypertriglyceridemia. The TT genotype of APOA5 c.553G ＞ T acts as an important indicator of hypertriglyceridemia in patients in Taiwan.

Peptide-based MRI contrast agent and near-infrared fluorescent probe for intratumoral legumain detection.

Recent studies suggest that intratumoral legumain promotes tumorigenesis. To monitor legumain activity in tumors, we developed a new MRI contrast agent ([Gd-NBCB-TTDA-Leg(L)]) and a NIR fluorescence probe (CyTE777-Leg(L)-CyTE807). The MRI contrast agent was prepared by introduction of cyclobutyl and benzyl group residues to TTDA (3,6,10-tri(carboxymethyl)-3,6,10-triaza-dodecanedioic acid), followed by the attachment of a legumain-specific substrate peptide (Leg(L)). The NIR fluorescence probe was designed by conjugating two NIR fluorochromes (CyTE777 and CyTE807) with Leg(L). Peptide cleavage of the MRI contrast agent by legumain can increase its hydrophobicity and promote rotational correlation time (τ(R)). Peptide cleavage of the NIR probes by the legumain relieves the self quench of the probe. Peptide cleavage of the MRI contrast agent and the NIR fluorescence probe by legumain were confirmed by T1 relaxometric studies and by fluorescence studies, respectively. In vivo MR images showed that [Gd-NBCB-TTDA-Leg(L)] attained 55.3 fold (254.2% versus 4.6%, at 2.0 h post-injection) higher imaging enhancement, as compared with control contrast agent bearing a noncleaveable peptide ([Gd-NBCB-TTDA-Leg(D)], in the CT-26 (legumain(+)) tumors. Similarly, optical imaging probe CyTE777-Leg(L)-CyTE807 attained 15.2 fold (3.34 × 10(9) photons/min versus 0.22 × 10(9) photons/min, at 24.0 h post-injection) higher imaging enhancement in the CT-26 (legumain(+)) tumors, compared to a NIR control probe (CyTE777-Leg(D)-CyTE807). These data indicate that the [Gd-NBCB-TTDA-Leg(L)] and the CyTE777-Leg(L)-CyTE807 probes may be promising tools to image the legumain-expressing cancers for diagnoses and targeted treatments.

Viral IRES prediction system - a web server for prediction of the IRES secondary structure in silico.

The internal ribosomal entry site (IRES) functions as cap-independent translation initiation sites in eukaryotic cells. IRES elements have been applied as useful tools for bi-cistronic expression vectors. Current RNA structure prediction programs are unable to predict precisely the potential IRES element. We have designed a viral IRES prediction system (VIPS) to perform the IRES secondary structure prediction. In order to obtain better results for the IRES prediction, the VIPS can evaluate and predict for all four different groups of IRESs with a higher accuracy. RNA secondary structure prediction, comparison, and pseudoknot prediction programs were implemented to form the three-stage procedure for the VIPS. The backbone of VIPS includes: the RNAL fold program, aimed to predict local RNA secondary structures by minimum free energy method; the RNA Align program, intended to compare predicted structures; and pknotsRG program, used to calculate the pseudoknot structure. VIPS was evaluated by using UTR database, IRES database and Virus database, and the accuracy rate of VIPS was assessed as 98.53%, 90.80%, 82.36% and 80.41% for IRES groups 1, 2, 3, and 4, respectively. This advance useful search approach for IRES structures will facilitate IRES related studies. The VIPS on-line website service is available at http://140.135.61.250/vips/.

Biochemical and genetic insights into asukamycin biosynthesis.

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled, and expressed heterologously in Streptomyces lividans. Bioinformatic analysis and mutagenesis studies elucidated the biosynthetic pathway at the genetic and biochemical level. Four gene sets, asuA-D, govern the formation and assembly of the asukamycin building blocks: a 3-amino-4-hydroxybenzoic acid core component, a cyclohexane ring, two triene polyketide chains, and a 2-amino-3-hydroxycyclopent-2-enone moiety to form the intermediate protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of protoasukamycin to 4-hydroxyprotoasukamycin, which is epoxidized at C5-C6 by AsuE3 to the final product, asukamycin. Branched acyl CoA starter units, derived from Val, Leu, and Ile, can be incorporated by the actions of the polyketide synthase III (KSIII) AsuC3/C4 as well as the cellular fatty acid synthase FabH to produce the asukamycin congeners A2-A7. In addition, the type II thioesterase AsuC15 limits the cellular level of omega-cyclohexyl fatty acids and likely maintains homeostasis of the cellular membrane.

Characterization of human papillomavirus infection in north Taiwan.

The detection of human papillomavirus (HPV) is very important for the evaluation of preventative strategies for cervical cancer. The major objective of this study was to characterize the prevalence of different genotypes of HPV in north Taiwan to contribute to the epidemiological knowledge of HPV infections. Papanicolaou (Pap) cervical smears were collected from 10,543 women aged between 14 and 87 years. The polymerase chain reaction (PCR) and DNA array hybridization techniques were used to genotype 51 different HPV strains. HPV was detected in 1,577 women, which gave an overall HPV prevalence rate of 15%. Forty-eight different genotypes were found in these patients, which included 9.7% that were high-risk HPV (HR-HPV) genotypes. The most common types of HR-HPV in patients, in descending order of frequency, were HPV 52, 16, 58, 56, 39, 51, 18, 68, 31, 33, 59, 45, and 35. HPV 52 was the most frequent type in every age group. The four most common HR-HPV types were found in 56.6% of the patients infected with HR-HPV. In cases that were infected with multiple HPV genotypes, 69.2% had at least one HR-HPV genotype. The rate of infection with HR-HPV was higher in the younger age groups than the older ones. In conclusion, 48 HPV genotypes were identified from a large study of cervical screening samples and the prevalence of HPV genotypes in different age groups was very diverse. The formulation of a public health strategy for HPV vaccination should take into account the prevalence of various HR-HPV/LR-HPV genotypes.

Nuclear-targeted inhibition of NF-kappaB on MMP-9 production by N-2-(4-bromophenyl) ethyl caffeamide in human monocytic cells.

Aberrant remodeling of the extracellular matrix occurs in many pathological processes, and its breakdown is mainly accomplished by matrix metalloproteinases (MMPs), which participate in the course of inflammation and tumor invasion. Nuclear factor-kappaB (NF-kappaB), a key transcription factor for the production of MMP-9, can be activated by various proinflammatory cytokines and promotes inflammation. In the present study, we investigated the intracellular mechanism for the inhibitory effects of an analogue of N-hydroxycinnamoylphenalkylamides, N-2-(4-bromophenyl) ethyl caffeamide (EK5), on tumor necrosis factor (TNF)-alpha stimulated expression of MMP-9 in a human monocytic cell line, THP-1. Our results show that TNF-alpha-induced expression of MMP-9 at both mRNA and protein levels was completely blocked by EK5 in a concentration-dependent (1-20microM) manner. We also found that EK5 markedly suppressed NF-kappaB signaling as detected by the NF-kappaB reporter gene assay but had no effects on the degradation of IkappaBalpha or translocation of NF-kappaB. Interestingly, chromatin immunoprecipitation results revealed that the association between p65 and MMP-9 promoter gene was completely abrogated by EK5, but the p65 phosphorylation was not affected. Overall, our findings suggest that EK5 inhibits MMP-9 production through the nuclear-targeted down-regulation of NF-kappaB signaling in human monocytic cells and this may provide a novel molecular basis of EK5 activity. Further studies are needed to verify its anti-inflammatory effects.

IRSS: a web-based tool for automatic layout and analysis of IRES secondary structure prediction and searching system in silico.

BACKGROUND: Internal ribosomal entry sites (IRESs) provide alternative, cap-independent translation initiation sites in eukaryotic cells. IRES elements are important factors in viral genomes and are also useful tools for bi-cistronic expression vectors. Most existing RNA structure prediction programs are unable to deal with IRES elements. RESULTS: We designed an IRES search system, named IRSS, to obtain better results for IRES prediction. RNA secondary structure prediction and comparison software programs were implemented to construct our two-stage strategy for the IRSS. Two software programs formed the backbone of IRSS: the RNAL fold program, used to predict local RNA secondary structures by minimum free energy method; and the RNA Align program, used to compare predicted structures. After complete viral genome database search, the IRSS have low error rate and up to 72.3% sensitivity in appropriated parameters. CONCLUSION: IRSS is freely available at this website http://140.135.61.9/ires/. In addition, all source codes, precompiled binaries, examples and documentations are downloadable for local execution. This new search approach for IRES elements will provide a useful research tool on IRES related studies.

The 5' untranslated region of Perina nuda virus (PnV) possesses a strong internal translation activity in baculovirus-infected insect cells.

A bicistronic baculovirus expression vector and fluorescent protein-based assays were used to identify the sequences that possess internal translation activity in baculovirus-infected insect cells. We demonstrated that the 5' untranslated region (5'UTR; 473 nucleotides) of Perina nuda virus (PnV) and the 5'UTR (579 nucleotides) of Rhopalosiphum padi virus (RhPV), but not the IRES sequence of Cricket paralysis virus, have internal translation activity in baculovirus-infected Sf21 cells. In addition, we found that including the first 22 codons of the predicted PnV open reading frame (ORF; a total of 539 nucleotides) enhanced internal translation activity by approximately 18 times. This is the first report of internal translation activity for a baculovirus expression system (BEVS) in the iflavirus 5' sequence and may facilitate the development of polycistronic baculovirus transfer vectors that can be used in BEVS for the production of multiple protein complexes.

Genomic shotgun array: a procedure linking large-scale DNA sequencing with regional transcript mapping.

To facilitate transcript mapping and to investigate alterations in genomic structure and gene expression in a defined genomic target, we developed a novel microarray-based method to detect transcriptional activity of the human chromosome 4q22-24 region. Loss of heterozygosity of human 4q22-24 is frequently observed in hepatocellular carcinoma (HCC). One hundred and eighteen well-characterized genes have been identified from this region. We took previously sequenced shotgun subclones as templates to amplify overlapping sequences for the genomic segment and constructed a chromosome-region-specific microarray. Using genomic DNA fragments as probes, we detected transcriptional activity from within this region among five different tissues. The hybridization results indicate that there are new transcripts that have not yet been identified by other methods. The existence of new transcripts encoded by genes in this region was confirmed by PCR cloning or cDNA library screening. The procedure reported here allows coupling of shotgun sequencing with transcript mapping and, potentially, detailed analysis of gene expression and chromosomal copy of the genomic sequence for the putative HCC tumor suppressor gene(s) in the 4q candidate region.

TDPOZ, a family of bipartite animal and plant proteins that contain the TRAF (TD) and POZ/BTB domains.

We have previously reported a gene Tdpoz1 (previously called 2cpoz56) that is temporally expressed in unfertilized eggs and in early embryos of the mouse. The putative TDPOZ1 protein carries a tumor necrosis factor receptor-associated factor (TRAF) domain (TD) and a POZ/BTB domain. On the analysis of nine bacterial artificial chromosome (BAC) clones, we have uncovered four more Tdpoz1 homologs in the mouse genome, designated Tdpoz2 through Tdpoz5. Tdpoz1 and Tdpoz2 are found 30 kb apart in a fully sequenced BAC clone (GenBank accession number AF545858). The genes are intronless in the coding region and each carries an intron in the 5'-untranslated region as in other early embryonic genes. The Tdpoz gene cluster is mapped on chromosome 3 at 3F2.1-2.2. RT-PCR experiments and a search of expressed sequence tag (EST) databases show that the Tdpoz1-5 genes are transcribed in early embryos, particularly at the two-cell stage. Exhaustive database searches have further uncovered three more mouse Tdpoz homologs in chromosomes 3 and 11 and 25 other Tdpoz-like orthologs in the genomes of other animal and plant species including human, rat, C. elegans, Drosophila, Arabidopsis and rice. In the rat genome, eight rat Tdpoz genes are found as a cluster in chromosome 2. Hence, TDPOZ proteins form a new protein family on the basis of similar protein domain organization. Based on reported characteristics of known TD- and POZ-bearing proteins, we speculate that TDPOZ proteins may be nuclear scaffold proteins probably involved in transcription regulation in early development and other cellular processes.

Comparative genome analysis of Vibrio vulnificus, a marine pathogen.

The halophile Vibrio vulnificus is an etiologic agent of human mortality from seafood-borne infections. We applied whole-genome sequencing and comparative analysis to investigate the evolution of this pathogen. The genome of biotype 1 strain, V. vulnificus YJ016, was sequenced and includes two chromosomes of estimated 3377 kbp and 1857 kbp in size, and a plasmid of 48,508 bp. A super-integron (SI) was identified, and the SI region spans 139 kbp and contains 188 gene cassettes. In contrast to non-SI sequences, the captured gene cassettes are unique for any given Vibrio species and are highly variable among V. vulnificus strains. Multiple rearrangements were found when comparing the 5.3-Mbp V. vulnificus YJ016 genome and the 4.0-Mbp V. cholerae El Tor N16961 genome. The organization of gene clusters of capsular polysaccharide, iron metabolism, and RTX toxin showed distinct genetic features of V. vulnificus and V. cholerae. The content of the V. vulnificus genome contained gene duplications and evidence of horizontal transfer, allowing for genetic diversity and function in the marine environment. The genomic information obtained in this study can be applied to monitoring vibrio infections and identifying virulence genes in V. vulnificus.

Linear plasmid SLP2 of Streptomyces lividans is a composite replicon.

SLP2 is a 50 kb linear plasmid in Streptomyces lividans that contains short (44 bp) terminal inverted repeats and covalently bound terminal proteins. The nucleotide sequence of SLP2 was determined. The rightmost 15.4 kb sequence is identical to that of the host chromosome, including the Tn4811 sequence at the border, which is interrupted by an insertion sequence (IS) element in SLP2. Examination of the flanking target sequences of Tn4811 suggests a previous recombinational event there. The 43 putative protein coding sequences contained many involved in replication (including two terminal protein homologues), partitioning, conjugal transfer and intramycelial spread. The terminally located helicase-like gene ttrA was necessary for conjugal transfer. The two telomeres diverge significantly in primary sequence, while preserving similar secondary structures. Mini-linear plasmids containing these telomeres replicated in S. lividans using the chromosomally encoded terminal protein. In addition, two pseudotelomere sequences are present near the left telomere. The G+C content and GC or AT skew profiles exhibit complex distributions. These, plus the inferred recombination at the right arm, indicate that SLP2 has evolved through rounds of exchanges involving at least three replicons.

Candida albicans SSD1 can suppress multiple mutations in Saccharomyces cerevisiae.

The SSD1 gene of Saccharomyces encodes a 160 kDa cytoplasmic protein that can suppress mutations in a number of other genes. A functional homologue of SSD1 from the human pathogen Candida albicans was isolated on the basis of its ability to restore viability at the restrictive temperature in a Saccharomyces cerevisiae swi4 ssd1-d strain. The C. albicans gene, designated CaSSD1, encodes a 1262 aa protein which has 47% identity overall to S. cerevisiae SSD1 as well as significant identity to Schizosaccharomyces pombe dis3 and sts5 products. It is shown that CaSSD1 expression is constitutive through the mitotic cell cycle, which is consistent with a role for the protein in cell growth. CaSSD1 rescues the swi4ts defect in an ssd1-d background when expressed from its own promoter on a single-copy plasmid and under the same conditions can rescue mutations in genes encoding protein phosphatase type 2A catalytic subunits. These data suggest that CaSSD1, like its S. cerevisiae homologue, can limit the effect of mutations on a variety of cellular processes.