Purification, sequencing and biological activity of polypeptide from velvet antler

The velvet antler polypeptide CNT14 was extracted and purified by gel filtration, ion exchange chromatography and RP C, which showed a single peak in HPLC chromatography and a single band in SDS-PAGE. The molecular weight measured by MALDI/TOF/MS spectrum was 1479. 9028. The polypeptide consisted mostly of Glu, Leu, Val, Pro. The amino acid sequence of the polypeptide was detected with ESI-MS/ MS, and the sequence was E-P-T-V-L-D-E-V-C-L-A-H-G-P. The experiments of biological activity of polypeptide CNT14 in vivo were carried out, and the results show that CNT14 has stimulant effects on the growth of rat HT22 cells. Then we produced the polypeptide CNT14 according the amino acid sequence by solid phase synthesis, confirmed the sequence of the polypeptide to be consistent with the amino acid sequence of polypeptide CNT14 which was separated from the velvet antler.

Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis

The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5'- and 3'-untranslated region(5'- and 3'-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1-547 bp, but they did not exist in the region of 548-2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.

Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis (vol 14, pg 307, 2002)

The geneswere cloned for the two apoprotein subunits, alpha and beta, of phycocyanin from the cyanobacterium Spirulina maxima (=Arthrospira maxima) strain F3. The alpha- and beta-subunit gene-coding regions contain 489 bp and 519 bp, respectively. The beta-subunit gene is upstream from the alpha-subunit gene, with a 111-bp segment separating them. Similarities between the alpha-subunits of S. maxima and nine other cyanobacteria were between 58% and 99%, as were those between the beta-subunits. The maximum similarity between the alpha- and beta-subunits from S. maxima was 27%.

Chiropteran types I and II interferon genes inferred from genome sequencing traces by a statistical gene-family assembler.

BACKGROUND: The rate of emergence of human pathogens is steadily increasing; most of these novel agents originate in wildlife. Bats, remarkably, are the natural reservoirs of many of the most pathogenic viruses in humans. There are two bat genome projects currently underway, a circumstance that promises to speed the discovery host factors important in the coevolution of bats with their viruses. These genomes, however, are not yet assembled and one of them will provide only low coverage, making the inference of most genes of immunological interest error-prone. Many more wildlife genome projects are underway and intend to provide only shallow coverage. RESULTS: We have developed a statistical method for the assembly of gene families from partial genomes. The method takes full advantage of the quality scores generated by base-calling software, incorporating them into a complete probabilistic error model, to overcome the limitation inherent in the inference of gene family members from partial sequence information. We validated the method by inferring the human IFNA genes from the genome trace archives, and used it to infer 61 type-I interferon genes, and single type-II interferon genes in the bats Pteropus vampyrus and Myotis lucifugus. We confirmed our inferences by direct cloning and sequencing of IFNA, IFNB, IFND, and IFNK in P. vampyrus, and by demonstrating transcription of some of the inferred genes by known interferon-inducing stimuli. CONCLUSION: The statistical trace assembler described here provides a reliable method for extracting information from the many available and forthcoming partial or shallow genome sequencing projects, thereby facilitating the study of a wider variety of organisms with ecological and biomedical significance to humans than would otherwise be possible.

Transmission of single HIV-1 genomes and dynamics of early immune escape revealed by ultra-deep sequencing.

We used ultra-deep sequencing to obtain tens of thousands of HIV-1 sequences from regions targeted by CD8+ T lymphocytes from longitudinal samples from three acutely infected subjects, and modeled viral evolution during the critical first weeks of infection. Previous studies suggested that a single virus established productive infection, but these conclusions were tempered because of limited sampling; now, we have greatly increased our confidence in this observation through modeling the observed earliest sample diversity based on vastly more extensive sampling. Conventional sequencing of HIV-1 from acute/early infection has shown different patterns of escape at different epitopes; we investigated the earliest escapes in exquisite detail. Over 3-6 weeks, ultradeep sequencing revealed that the virus explored an extraordinary array of potential escape routes in the process of evading the earliest CD8 T-lymphocyte responses--using 454 sequencing, we identified over 50 variant forms of each targeted epitope during early immune escape, while only 2-7 variants were detected in the same samples via conventional sequencing. In contrast to the diversity seen within epitopes, non-epitope regions, including the Envelope V3 region, which was sequenced as a control in each subject, displayed very low levels of variation. In early infection, in the regions sequenced, the consensus forms did not have a fitness advantage large enough to trigger reversion to consensus amino acids in the absence of immune pressure. In one subject, a genetic bottleneck was observed, with extensive diversity at the second time point narrowing to two dominant escape forms by the third time point, all within two months of infection. Traces of immune escape were observed in the earliest samples, suggesting that immune pressure is present and effective earlier than previously reported; quantifying the loss rate of the founder virus suggests a direct role for CD8 T-lymphocyte responses in viral containment after peak viremia. Dramatic shifts in the frequencies of epitope variants during the first weeks of infection revealed a complex interplay between viral fitness and immune escape.

Elucidation of hepatitis C virus transmission and early diversification by single genome sequencing.

A precise molecular identification of transmitted hepatitis C virus (HCV) genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of 2,922 half or quarter genomes from plasma viral RNA to identify transmitted/founder (T/F) viruses in 17 subjects with acute community-acquired HCV infection. Sequences from 13 of 17 acute subjects, but none of 14 chronic controls, exhibited one or more discrete low diversity viral lineages. Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes. Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more (median = 4). Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny. In these cases, empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy. These results provide new quantitative and qualitative insights into HCV transmission, revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome. Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures.

Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum-associated degradation pathway.

PURPOSE: The endoplasmic reticulum-associated degradation pathway is responsible for the translocation of misfolded proteins across the endoplasmic reticulum membrane into the cytosol for subsequent degradation by the proteasome. To define the phenotype associated with a novel inherited disorder of cytosolic endoplasmic reticulum-associated degradation pathway dysfunction, we studied a series of eight patients with deficiency of N-glycanase 1. METHODS: Whole-genome, whole-exome, or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data. RESULTS: All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypolacrima or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele. CONCLUSION: NGLY1 deficiency is a novel autosomal recessive disorder of the endoplasmic reticulum-associated degradation pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a broader range of mutations are detected.

Hybrid error correction and de novo assembly of single-molecule sequencing reads.

Single-molecule sequencing instruments can generate multikilobase sequences with the potential to greatly improve genome and transcriptome assembly. However, the error rates of single-molecule reads are high, which has limited their use thus far to resequencing bacteria. To address this limitation, we introduce a correction algorithm and assembly strategy that uses short, high-fidelity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on reads generated by a PacBio RS instrument from phage, prokaryotic and eukaryotic whole genomes, including the previously unsequenced genome of the parrot Melopsittacus undulatus, as well as for RNA-Seq reads of the corn (Zea mays) transcriptome. Our long-read correction achieves >99.9% base-call accuracy, leading to substantially better assemblies than current sequencing strategies: in the best example, the median contig size was quintupled relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes.

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

Rare hereditary COL4A3/COL4A4 variants may be mistaken for familial focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a histological lesion with many causes, including inherited genetic defects, with significant proteinuria being the predominant clinical finding at presentation. Mutations in COL4A3 and COL4A4 are known to cause Alport syndrome (AS), thin basement membrane nephropathy, and to result in pathognomonic glomerular basement membrane (GBM) findings. Secondary FSGS is known to develop in classic AS at later stages of the disease. Here, we present seven families with rare or novel variants in COL4A3 or COL4A4 (six with single and one with two heterozygous variants) from a cohort of 70 families with a diagnosis of hereditary FSGS. The predominant clinical finding at diagnosis was proteinuria associated with hematuria. In all seven families, there were individuals with nephrotic-range proteinuria with histologic features of FSGS by light microscopy. In one family, electron microscopy showed thin GBM, but four other families had variable findings inconsistent with classical Alport nephritis. There was no recurrence of disease after kidney transplantation. Families with COL4A3 and COL4A4 variants that segregated with disease represent 10% of our cohort. Thus, COL4A3 and COL4A4 variants should be considered in the interpretation of next-generation sequencing data from such patients. Furthermore, this study illustrates the power of molecular genetic diagnostics in the clarification of renal phenotypes.

High-coverage sequencing and annotated assemblies of the budgerigar genome.

BACKGROUND: Parrots belong to a group of behaviorally advanced vertebrates and have an advanced ability of vocal learning relative to other vocal-learning birds. They can imitate human speech, synchronize their body movements to a rhythmic beat, and understand complex concepts of referential meaning to sounds. However, little is known about the genetics of these traits. Elucidating the genetic bases would require whole genome sequencing and a robust assembly of a parrot genome. FINDINGS: We present a genomic resource for the budgerigar, an Australian Parakeet (Melopsittacus undulatus) -- the most widely studied parrot species in neuroscience and behavior. We present genomic sequence data that includes over 300× raw read coverage from multiple sequencing technologies and chromosome optical maps from a single male animal. The reads and optical maps were used to create three hybrid assemblies representing some of the largest genomic scaffolds to date for a bird; two of which were annotated based on similarities to reference sets of non-redundant human, zebra finch and chicken proteins, and budgerigar transcriptome sequence assemblies. The sequence reads for this project were in part generated and used for both the Assemblathon 2 competition and the first de novo assembly of a giga-scale vertebrate genome utilizing PacBio single-molecule sequencing. CONCLUSIONS: Across several quality metrics, these budgerigar assemblies are comparable to or better than the chicken and zebra finch genome assemblies built from traditional Sanger sequencing reads, and are sufficient to analyze regions that are difficult to sequence and assemble, including those not yet assembled in prior bird genomes, and promoter regions of genes differentially regulated in vocal learning brain regions. This work provides valuable data and material for genome technology development and for investigating the genomics of complex behavioral traits.

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.