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At least 145 records · Page 8Linked to original sources

PubMed2002

Search for potential vaccine candidate open reading frames in the Bacillus anthracis virulence plasmid pXO1: in silico and in vitro screening.

A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis, Bacillus halodurans, and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from approximately 30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.

Animals↗

PubMed2025

Comprehensive profiling of lysine lactylation in Candida albicans and exploratory analysis of fluconazole tolerance associations.

UNLABELLED: Candida albicans is the primary pathogen of invasive candidiasis in most regions worldwide, but the therapy options for C. albicans infections are limited, and drug tolerance further exacerbates the treatment challenges. Lysine lactylation (Kla), a recently identified post-translational modification (PTM), is observed in numerous organisms; however, the role of Kla in C. albicans remains unknown. Hence, we report the first proteomic analysis of this specific modification in C. albicans and discuss its potential roles in drug tolerance of C. albicans. Altogether, 7,233 lactylation sites on 1,608 lactylated proteins were identified in C. albicans, with the highest degree of lactylation among the species studied so far. The further bioinformatics analysis revealed that the lactylated proteins were implicated in a variety of cellular functions with diverse subcellular localizations. Additionally, we found a unique survival mode of tolerant cells in the presence of fluconazole, which will be subject to a more thorough investigation in our future studies. This paper is the first report on the lactylome of Candida spp. and provides a reliable foundation for further research on Kla in C. albicans and other human pathogens. IMPORTANCE: This is the first report on the lactylome of Candida spp., and it provides some valuable insights for further research on lactylation in C. albicans and other human pathogens. Moreover, the observations in tolerant cells have prompted plausible hypotheses regarding the potential role of lactylation in mediating C. albicans tolerance to fluconazole, thereby offering a conceptual framework for subsequent investigations. Notably, fungal tolerance to azoles, a concept distinct from resistance, represents a critical phenomenon in C. albicans with profound clinical implications, as it directly correlates with therapeutic failure and persistent infections.

Candida albicans↗

PubMed2026

Proteomic insights into azoospermia: protein differences in testicular tissue between non-obstructive and obstructive azoospermia patients.

Non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) are the main classifications of severe male infertility, but the molecular mechanism of NOA remains poorly understood. This study aimed to identify potential biomarkers and pathological mechanisms by comparing the proteomic differences in testicular tissues of NOA and OA patients. Through proteomic analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of testicular samples from 5 NOA patients and 5 OA patients, we identified 5264 proteins, among which 717 differentially expressed proteins (DEPs) were found between the two groups (242 upregulated and 475 downregulated in NOA). Bioinformatics analysis indicated that these DEPs were significantly associated with reproductive development, gametogenesis, and cell structural stability. On the basis of this, six candidate proteins, including dysferlin (DYSF), myoferlin (MYOF), mitsugumin 53 (MG53), cluster of differentiation 63 (CD63), caveolin-3 (CAV3), and calpain-3 (CAPN3), were selected from the DEPs and verified in an expanded sample set (37 NOA cases and 28 OA cases) through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, confirming their dysregulation in NOA. These findings provide new proteomic insights into NOA, highlighting the disruption of membrane repair and structural pathways, and offer potential biomarkers for understanding its pathogenesis.

Humans↗

PubMed2001

Molecular cloning, structure and expression of a novel nuclear RNA-binding cyclophilin-like gene (PPIL4) from human fetal brain.

The cyclophilins are members of a highly conserved, ubiquitous family, and play an important role in protein folding, immunosuppression by cyclosporin A (CsA), and infection of HIV-1 virions. Here we report that a novel member of the cyclophilin family, PPIL4, was cloned and identified during the large-scale sequencing analysis from a human fetal brain cDNA library. The PPIL4 gene encodes a protein which shares 96% amino acid identity with a protein encoded by a putative gene recently cloned from several different early stages of mouse embryo, and its homologues are found in several other organisms. According to bioinformatics analysis, the PPIL4 gene was found to be located in chromosome 6q24-->q25. Besides the PPIase motif, PPIL4 also has an RNA recognition motif (RRM), a pair of bipartite nuclear targeting sequences, and a lysine rich domain. RT-PCR analysis indicated that PPIL4 gene expression is abundant in kidney but has a ubiquitously low expression pattern in other human adult tissues.

Amino Acid Motifs↗

PubMed2026

[Study of a patient with azoospermia due to variant of MOV10L1 gene].

OBJECTIVE: To explore the clinical and genotypic characteristics of a patient with Sertoli cell-only syndrome (SCOS) due to variants of MOV10L1 gene. METHODS: A 27-year-old patient with Non-obstructive azoospermia (NOA) underwent routine semen analysis. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P), estradiol (E2), prolactin (PRL), and testosterone (T) were determined by chemiluminescence assays. Peripheral blood samples were collected for G-banded karyotyping analysis. Multiplex PCR fluorescence detection was used to screen for AZF gene microdeletions. Whole exome sequencing (WES) and Sanger sequencing were performed simultaneously. Testicular biopsy tissues were subjected to Hematoxylin-Eosin (HE) staining to assess seminiferous tubule cell composition, and MOV10L1 protein expression was detected by immunohistochemical staining. Bioinformatics tools were employed to predict the pathogenicity of variants and their impact on protein structure and function. This study was approved by the Medical Ethics Committee of the Guangdong Institute of Reproductive Sciences [Ethics No.: 2023(01)]. RESULTS: The patient's two semen analyses had failed to detect any sperm. Hormone tests indicated elevated FSH (22.32 mIU/mL) and PRL (397.6 mIU/mL), while T (3.68 nmol/L) and E2 (38.32 pmol/L) were reduced. Chromosomal karyotyping revealed 46,XY, and no AZF gene deletion was detected. WES and Sanger sequencing detected compound heterozygous variants of the MOV10L1 gene, including a c.345C>A (p.C115X) nonsense variant and a c.3323C>T (p.T1108I) missense variant, with the former being unreported previously. HE staining showed only Sertoli cells in the seminiferous tubules, confirming the diagnosis of SCOS. Immunohistochemical staining revealed absent MOV10L1 protein expression in the testicular tissue. Based on the guidelines from American College of Medical Genetics and Genomics (ACMG), the c.345C>A (p.C115X) was classified as a pathogenic variant (PVS1+PM2_Supporting+PP4), while the c.3323C>T (p.T1108I) was deemed variant of uncertain significance (PM2_Supporting+PP3_Supporting+PP4). Bioinformatics analysis demonstrated that c.345C>A (p.C115X) may cause premature termination of protein translation, while c.3323C>T (p.T1108I) may disrupt the hydrophobicity of the RNA helicase domain, reducing the active pocket volume and decreasing its affinity for MILI protein. CONCLUSION: This study has diagnosed a case of SCOS due to compound heterozygous variants of the MOV10L1 gene, which also enriched its mutational spectrum.

Humans↗

PubMed2003

Screening and identification of the up-regulated genes in human mesangial cells exposed to angiotensin II.

Accumulation of extracellular matrix (ECM) in the glomerular mesangium is a common feature of many progressive renal diseases. Angiotensin II (Ang II) plays a major role in the progression of chronic kidney diseases in part by induction of ECM. However, the precise molecular signals responsible for this effect are unknown. To explore possible molecular mechanisms of ECM production related to Ang II, we screened and identified genes up-regulated by Ang II in cultured human mesangial cells (MC). Detection of up-regulated genes was determined by mRNA populations from human MC with and without Ang II stimulation (10(-6) mol/l, 24 h) by suppression subtractive hybridization. Reverse Northern blot analysis was performed to screen for differentially expressed genes. Full-length cDNAs of three novel genes were isolated by rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR). One of these novel genes, AngRem104, was further investigated by Northern blot, Western blot and reverse transcription (RT)-PCR. The bioinformatics analysis implied that AngRem104 coded for a nuclear protein that was widely expressed in various normal human tissues. Moreover, up-regulation of AngRem104 induced by Ang II was time-dependent and was dose-dependently blocked by the Ang II type 1 receptor antagonist, Losartan. Interestingly, we also demonstrated that AngReam104 was associated with increased fibronectin expression. We conclude that AngRem104 is a novel human gene that is related to the expression of fibronectin and that is up-regulated by Ang II in human MC. These findings may lead to new insights into the mechanisms of glomerular sclerosis associated with Ang II.

Amino Acid Sequence↗

PubMed2002

The promoter analysis of the human C17orf25 gene, a novel chromosome 17p13.3 gene.

The human C17orf25 gene (Accession No. AF177342) is one of thirteen genes cloned from a region displaying a high score of loss of heterozygosity within chromosome 17p13.3 in human hepatocellular carcinoma in China. To unveil the underlying mechanisms for the transcription regulation of this gene and understand its implication to the hepatocellular carcinogenesis, we looked into the relevant aspects by both bioinformatic and experimental executions. We found: 1, The abundant expression of the C17orf25 gene was evident in all the cell lines and tissue samples tested, showing little hepatoma-selectivity; 2, Its transcription starts at a single site, locating at -60 from the translation initiation codon; 3, A 58 bp fragment containing the transcription start, extending from -112 to -55, represents the minimal promoter; 4, The consensus sequence within this fragment recognized by SP1 contributes predominantly to the activity of the minimal promoter; 5, The bioinformatic analysis suggests that the C17orf25 gene may encode a protein in the family of the glyoxalase. Our data has provided some deep insight into both function and regulation of the C17orf25 gene in the context of the normal liver and hepatocellular carcinoma.

5' Flanking Region↗

PubMed2026

Mechanic evaluation of Jisheng Shenqi Wan on calcium oxalate kidney stones: an integrated network pharmacology and metabolomics.

OBJECTIVE: To understand the efficacy of Jisheng Shenqi Wan (JSSQW, ) in treating calcium oxalate kidney stones (KS) and to investigate the mechanism of JSSQW action by combining network pharmacology with metabolomics analysis based on ultra-high performance liquid chromatography combined with tandem electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS). METHODS: The chemical components of JSSQW absorbed into rat blood were identified by UHPLC-Q/Orbitrap HRMS. The identified components were introduced into the Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine platform to screen for target genes, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and disease enrichment analysis. A KS rat model was generated using the oxalic acid precursor method to examine the efficacy of JSSQW for treating KS. Serum metabolomics was used to monitor changes in endogenous substances in KS rats after JSSQW intervention. RESULTS: Twenty-three chemicals from JSSQW were identified in the blood of JSSQW gavage-administered rats. KEGG enrichment analysis predicted the top 20 signaling pathways affected by these 23 chemicals. Disease enrichment analysis showed that the target genes of these 23 chemicals were enriched in diseases of the urinary system and endocrine system, including kidney stones. In a KS rat model, JSSQW inhibited the aggregation of calcium oxalate crystals, reduced renal tubular injury, lowered the renal index, and improved biochemical indicators (blood creatinine, blood urea nitrogen). Serum metabolomics identified 25 differential metabolites that responded to JSSQW treatment. They were mainly lipids, with phosphatidylethanolamine and phosphorylcholine and their derivatives accounting for the highest proportion. Metabolic pathway analysis showed that the changes in differential metabolites were related to multiple metabolic pathways, especially sphingolipid metabolism and sphingolipid signaling pathways. CONCLUSIONS: JSSQW can inhibit the aggregation of calcium oxalate crystals in the kidneys, reduce tubular injury, and improve kidney function in KS rats. Its mechanism of action may be related to regulating disordered metabolites and metabolic pathways, especially glycerol phospholipid metabolism, sphingolipid metabolism, and sphingolipid signaling.

Drugs, Chinese Herbal↗

PubMed2025

Investigating the miRNA-mRNA interactome of human trabecular meshwork cells treated with TGF-β1 provides insights into the pathogenesis of pseudoexfoliation glaucoma.

Pseudoexfoliation glaucoma is a severe form of secondary open angle glaucoma and is associated with activation of the TGF-β pathway by TGF-β1. MicroRNAs (miRNAs) are small non-coding RNA species that are involved in regulation of mRNA expression and translation. To investigate what glaucomatous changes occur in the trabecular meshwork and how these changes may be regulated by miRNAs, we performed a bioinformatics analysis resulting in a miRNA-mRNA interactome. Primary human trabecular meshwork cells originating from normal donors were treated with TGF-β1 at 5 ng/mL for 24h; total RNA was extracted followed by RNA-Seq and miRNA-Seq. For both mRNA and miRNA species, differential expression was determined using a bioinformatics pipeline consisting of FastQC, STAR, FeatureCounts, edgeR (for miRNA) and DESeq2 (for mRNA). Putative mRNA-miRNA interactions between differentially expressed mRNA and miRNA species were determined using interaction databases miRWalk, miRTarBase, TarBase and TargetScan. To classify mRNA species by function and pathway, gene enrichment was performed using Enrichr. The resulting miRNA-mRNA interactome consisted of 1202 interactions. Some highly connected microRNAs were hsa-let-7e-5p, hsa-miR-20a-5p, hsa-miR-122-5p, and hsa-miR-29c-3p. Most differentially expressed genes were indicated to be regulated by miRNAs. The sub-interactomes of genes involved in specific pseudoexfoliation glaucoma related enrichment terms such as oxidative stress, unfolded protein response, signal molecules and ECM remodelling were determined. This is the first study to present a genome-wide microRNA-mRNA regulatory network for human trabecular meshwork cells treated with TGF-β1 and may serve to generate unbiased hypotheses about regulatory functions and mRNA targets of miRNAs in pseudoexfoliation glaucoma and may help to develop miRNA-based therapeutics.

Humans↗

PubMed2025

miR-9-5p/HMMR regulates the tumorigenesis and progression of clear cell renal cell carcinoma through EMT and JAK1/STAT1 signaling pathway.

BACKGROUND: The most common malignant type of kidney cancer is clear cell renal cell carcinoma (ccRCC). The expression levels of hyaluronan-mediated motility receptor (HMMR) in many tumor types are significantly elevated. HMMR is closely associated with tumor-related progression, treatment resistance, and poor prognosis, and has yet to be fully investigated in terms of its expression patterns and molecular mechanisms of action in ccRCC. Further research is imperative to elucidate these aspects. METHODS: We used The Cancer Genome Atlas (TCGA) database to preliminarily investigate HMMR expression and function in ccRCC and the data for 19 samples from the NCBI GEO database (GSE207493) for single-cell analysis. We assessed the differential expression level of HMMR between ccRCC cancerous tissues and their matched non-tumor tissues. Subsequently, a series of in vivo and in vitro experiments were designed to elucidate the biological function of HMMR in ccRCC, including Transwell assays, CCK-8 assays, clone formation assays and subcutaneous xenograft experiments in nude mice. Through bioinformatics analysis, we identified potential microRNAs (miRNAs) that may regulate HMMR, as well as the possible signaling pathways involved. Finally, we conducted a series of cellular functional experiments to validate our hypotheses regarding the HMMR axis. RESULTS: HMMR expression was significantly up-regulated in tumor tissues of ccRCC patients, and elevated HMMR expression level showed a strong correlation with ccRCC progression and adverse prognoses of patients. Knocking down HMMR inhibited the proliferative and migratory abilities of ccRCC cells, while its overexpression amplified these oncogenic properties. In nude mice model, reduced HMMR expression inhibited ccRCC tumor proliferation in vivo. Furthermore, overexpression of an upstream transcriptional regulator, miR-9-5p, effectively downregulated HMMR expression and thus impeded ccRCC cells proliferation and migration. HMMR might influence ccRCC growth via the Epithelial-Mesenchymal Transition (EMT) pathway and the Janus Kinase 1/Signal Transducer and Activator of Transcription 1 (JAK1/STAT1) pathway. CONCLUSIONS: HMMR is overexpressed in ccRCC, and there is a significant link between high HMMR expression and tumor progression, as well as poor patient prognosis. Specifically, HMMR could be targeted and inhibited by miR-9-5p and might modulate the tumorigenesis and progression of ccRCC through both EMT and JAK1/STAT1 signaling pathway.

Carcinoma, Renal Cell↗

PubMed2026

Hypertrophic cardiomyopathy: a genome-wide association meta-analysis and polygenic risk score.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a heritable trait with marked variability in expression and outcomes. Our aims were to discover new genetic loci associated with HCM and to test the effect of a new polygenic risk score (PRS) on incidence, phenotype and outcomes stratified by genotype status. METHODS: A discovery genome-wide association study (GWAS) was performed on 2284 HCM cases and 4525 controls. Two fixed-effects meta-analyses combined our discovery GWAS with single-trait and multi-trait results from a published study. Discovered loci underwent comprehensive bioinformatic analysis including functional and druggability annotations. A PRS using loci from the two meta-analyses was evaluated for association with HCM diagnosis in 411 213 individuals from UK Biobank (UKBB); imaging phenotypes in individuals without HCM; a composite endpoint (including all-cause mortality and transplantation); and sudden cardiac death (SCD) in 1756 HCM cases. PRS analyses were stratified by genotype status. RESULTS: Three loci were found in the discovery GWAS (BAG3, FHOD3 and novel locus PPP1R3A). In the meta-analyses, 70 unique loci were identified, four novel (MYPN, YWHAE, NOS1AP and OBSCN). Bioinformatic analyses identified NOS1AP as a candidate HCM gene. A new PRS was significantly associated with HCM diagnosis (HR=3.19, 95% CI 2.46 to 4.14 for top 5% vs lower 95%; HR=1.88, 95% CI 1.72 to 2.06 per SD increase). Significant associations were found between PRS and greater left ventricular (LV) wall thickness and higher LV ejection fraction in UKBB participants without HCM. Genotype-negative HCM cases in the top 20% of the PRS distribution had an increased risk of SCD (HR=2.72, 95% CI 1.03 to 7.17). CONCLUSIONS: We report novel HCM loci. A new PRS predicted the risk of HCM development and associated imaging characteristics in the UKBB and outcomes in an HCM cohort.

Cardiomyopathies↗

PubMed2026

A CHO-Derived Matrix Attachment Region Enhances Transgene Dosage, SATB1 Recruitment, and Monoclonal Antibody Expression in a Dual-Promoter Vector System.

The production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is often affected by position-effect variegation and the gradual loss of transgene expression over time. Hence, we have designed a dual-promoter IgG expression vector and compared versions that either contained or lacked a CHO-derived matrix-attachment region (MAR). Stable CHO-S pools, cultured in serum-free conditions, revealed that the MAR-containing construct produced higher and more consistent antibody levels across ten passages, as confirmed by Western blot and Protein A Octet analysis. Product-quality analysis by size-exclusion chromatography and reducing SDS-PAGE confirmed formation of properly assembled, mainly monomeric antibodies in both cases. Quantitative PCR indicated greater transgene copy numbers in MAR pools (+ 48% for the light chain and + 71% for the heavy chain), and RT-qPCR showed roughly fourfold higher transcript levels for both chains relative to controls. Bioinformatic analysis revealed several SATB1 binding motifs within the MAR sequence, and ChIP-qPCR demonstrated SATB1 association with the MAR-linked transgene locus. Overall, the data suggested that a CHO-native MAR could enhance transgene dosage and transcriptional activity, while preserving product integrity, possibly through SATB1-mediated chromatin organization. Ongoing work includes chromatin-mark profiling and process-level productivity measurements to better define the impact of MAR-based vector design on biomanufacturing performance.

Animals↗

PubMed2024

A novel splice-altering TNC variant (c.5247A > T, p.Gly1749Gly) in an Chinese family with autosomal dominant non-syndromic hearing loss.

BACKGROUND: This study aims to analyze the pathogenic gene in a Chinese family with non-syndromic hearing loss and identify a novel mutation site in the TNC gene. METHODS: A five-generation Chinese family from Anhui Province, presenting with autosomal dominant non-syndromic hearing loss, was recruited for this study. By analyzing the family history, conducting clinical examinations, and performing genetic analysis, we have thoroughly investigated potential pathogenic factors in this family. The peripheral blood samples were obtained from 20 family members, and the pathogenic genes were identified through whole exome sequencing. Subsequently, the mutation of gene locus was confirmed using Sanger sequencing. The conservation of TNC mutation sites was assessed using Clustal Omega software. We utilized functional prediction software including dbscSNV_AdaBoost, dbscSNV_RandomForest, NNSplice, NetGene2, and Mutation Taster to accurately predict the pathogenicity of these mutations. Furthermore, exon deletions were validated through RT-PCR analysis. RESULTS: The family exhibited autosomal dominant, progressive, post-lingual, non-syndromic hearing loss. A novel synonymous variant (c.5247A > T, p.Gly1749Gly) in TNC was identified in affected members. This variant is situated at the exon-intron junction boundary towards the end of exon 18. Notably, glycine residue at position 1749 is highly conserved across various species. Bioinformatics analysis indicates that this synonymous mutation leads to the disruption of the 5' end donor splicing site in the 18th intron of the TNC gene. Meanwhile, verification experiments have demonstrated that this synonymous mutation disrupts the splicing process of exon 18, leading to complete exon 18 skipping and direct splicing between exons 17 and 19. CONCLUSION: This novel splice-altering variant (c.5247A > T, p.Gly1749Gly) in exon 18 of the TNC gene disrupts normal gene splicing and causes hearing loss among HBD families.

Adult↗

PubMed2002

Structure prediction and phylogenetic analysis of a functionally diverse family of proteins homologous to the MT-A70 subunit of the human mRNA:m(6)A methyltransferase.

MT-A70 is the S-adenosylmethionine-binding subunit of human mRNA:m(6)A methyl-transferase (MTase), an enzyme that sequence-specifically methylates adenines in pre-mRNAs. The physiological importance yet limited understanding of MT-A70 and its apparent lack of similarity to other known RNA MTases combined to make this protein an attractive target for bioinformatic analysis. The sequence of MT-A70 was subjected to extensive in silico analysis to identify orthologous and paralogous polypeptides. This analysis revealed that the MT-A70 family comprises four subfamilies with varying degrees of interrelatedness. One subfamily is a small group of bacterial DNA:m(6)A MTases. The other three subfamilies are paralogous eukaryotic lineages, two of which have not been associated with MTase activity but include proteins having substantial regulatory effects. Multiple sequence alignments and structure prediction for members of all four subfamilies indicated a high probability that a consensus MTase fold domain is present. Significantly, this consensus fold shows the permuted topology characteristic of the b class of MTases, which to date has only been known to include DNA MTases.

Amino Acid Sequence↗

PubMedDate not supplied

Studies of quantitative analysis of protein expression in Saccharomyces cerevisiae.

In the present study amino acid analysis is applied to quantitation of Saccharomyces cerevisiae proteome expression. The quantitation levels obtained are compared to data using densitometric analysis of silver or amido black staining and to the theoretical expression level (codon bias) of the identified proteins determined from their amino acid analysis (AAA). The results show that relative volume ratio (%vol) using Melanie II is a better parameter for spot quantitation than relative optical density ratio (%OD), and amino black staining provides good linearity within the range 1-100 pmol protein. However, AAA shows that theoretical expression levels are not well correlated with actual protein expression level, although there is better correlation when isoforms of the expressed protein are identified and included. It is concluded that amino acid analysis provides accurate protein quantitation and has a continuing role in proteome studies in terms of the rapid and inexpensive quantitation of proteins displayed on proteome maps. We do however recognize that in the context of future clinical applications and large-scale proteome discovery projects, quantitation and post-translational modification need to be analyzed by 'proteomatic' (i.e., proteome automatic bioinformatic analysis directly from the gel) techniques.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2001

Identification of the human crooked neck gene by comparative gene identification.

Drosophila melanogaster serves as a useful model organism for functional genomic studies, and its genome project was recently completed. We previously described a comparative-gene-identification approach to assist human ortholog gene identification that involves applying an entire proteome as an alignment template. Analysis of the available 14100 Drosophila protein sequences revealed that 37% of them (5228 sequences) might lead to discoveries of novel human genes. Upon further database interrogations, we found several putative full-length human gene transcripts, including the human crooked neck (crn) gene. Based on sequence gap-closure experiments using reverse transcriptase-polymerase chain reaction as well as bioinformatic analysis, we found that the assembled human cDNA contig of crooked neck gene was at least 3903 base pairs in length with alternative splicing variations which encoded mainly for a 687-amino-acid residue protein. The human crooked neck gene was located on chromosome 20 with at least 15 exons. The unique features of the 16 copies of the tetratrico peptide repeat (TPR) motif were conserved in the yeast, fly and human crooked neck orthologous proteins, which were important for spliceosome assembly in cells.

Amino Acid Sequence↗

PubMed2002

Functional organization of the yeast proteome by systematic analysis of protein complexes.

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

Cells, Cultured↗

PubMed2002

Modular organization of phylogenetically conserved domains controlling developmental regulation of the human skeletal myosin heavy chain gene family.

The mammalian skeletal myosin heavy chain locus is composed of a six-membered family of tandemly linked genes whose complex regulation plays a central role in striated muscle development and diversification. We have used publicly available genomic DNA sequences to provide a theoretical foundation for an experimental analysis of transcriptional regulation among the six promoters at this locus. After reconstruction of annotated drafts of the human and murine loci from fragmented DNA sequences, phylogenetic footprint analysis of each of the six promoters using standard and Bayesian alignment algorithms revealed unexpected patterns of DNA sequence conservation among orthologous and paralogous gene pairs. The conserved domains within 2.0 kilobases of each transcriptional start site are rich in putative muscle-specific transcription factor binding sites. Experiments based on plasmid transfection in vitro and electroporation in vivo validated several predictions of the bioinformatic analysis, yielding a picture of synergistic interaction between proximal and distal promoter elements in controlling developmental stage-specific gene activation. Of particular interest for future studies of heterologous gene expression is a 650-base pair construct containing modules from the proximal and distal human embryonic myosin heavy chain promoter that drives extraordinarily powerful transcription during muscle differentiation in vitro.

Animals↗