Transcripts Of Unknown Function Research Articles

The dark proteome: translation from noncanonical open reading frames.

Omics-based technologies have revolutionized our understanding of the coding potential of the genome. In particular, these studies revealed widespread unannotated open reading frames (ORFs) throughout genomes and that these regions have the potential to encode novel functional (micro-)proteins and/or hold regulatory roles. However, despite their genomic prevalence, relatively few of these noncanonical ORFs have been functionally characterized, likely in part due to their under-recognition by the broader scientific community. The few that have been investigated in detail have demonstrated their essentiality in critical and divergent biological processes. As such, here we aim to discuss recent advances in understanding the diversity of noncanonical ORFs and their roles, as well as detail biologically important examples within the context of the mammalian genome.

Actinidia-Pseudomonas syringae pv. actinidiae interaction: differentially expressed plant transcripts during infection

Since 2008, kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has spread to pandemic scale. In order to elucidate the molecular events of the kiwifruit-Psa interaction, Illumina RNAseq was conducted after Psa inoculation and mock inoculation, on both acibenzolar-S-methyl (ASM) treated and untreated Actinidia chinensis plants. De-novo assembly of the kiwifruit transcriptome revealed 39,607 transcripts. To investigate the transcriptome expression profile during the infection process, hierarchical cluster analysis was performed. During the infection process, about 3000 transcripts were modulated. It was notable that the number of modulated transcripts in infected plants were higher than in ASM-treated infected plants. Furthermore, the RNAseq technology permitted us to identify novel transcripts of unknown function but related to infection and ASM treatment. The analysis revealed that the coordinates of some important gene functional categories change, such as hormonal balance, transcriptional regulation and signalling. Different transcription factors were also linked with signalling and hormonal balance and are involved in the ability of ASM-treated plants to assemble the right defence response against Psa. On the other hand, control plants respond to Psa, but not in an adequate way to counteract the infection. Indeed, several transcripts related to plant signalling and putatively encoding RLK-LRR and PRR proteins were exclusively found under these conditions, while they were absent in ASM-treated plants, highlighting how the response of susceptible plants belongs to a separate cluster from that of ASM-treated plants.

Micropeptides Encoded in Transcripts Previously Identified as Long Noncoding RNAs: A New Chapter in Transcriptomics and Proteomics.

Integrative analysis using omics-based technologies results in the identification of a large number of putative short open reading frames (sORFs) with protein-coding capacity within transcripts previously identified as long noncoding RNAs (lncRNAs) or transcripts of unknown function (TUFs). sORFs were previously overlooked because of their diminutive size and the difficulty of identification by bioinformatics analyses. There is now growing evidence of the existence of potentially functional micropeptides produced from sORFs within cells of diverse species. Recent characterization of a few of these revealed their significant divergent roles in many fundamental biological processes, where some also show important relationships with pathogenesis. Recent works therefore provide new insights for exploring the wealth of information that may lie within sORF-encoded short proteins. Here, we summarize the current progress and view of micropeptides encoded in sORFs of protein-coding genes.

OR23 Elucidating the targetome of the HLA-B intron 4 derived miRNA, miR-6891 and allele specific miRNA isoforms

Aim Next generation sequencing of HLA loci facilitates the full characterization of HLA alleles at an unprecedented rate. However, little is known about the influence of HLA intronic sequence variation in the pathophysiology of transplant medicine or immune-mediated disease. Exploration of HLA intronic loci reveals that an annotated miRNA hairpin, miR-6891 is derived from intron 4 of HLA-B (Fig. 1), giving rise to two mature miRNA transcripts of unknown function. RNA-seq data reveals that miR-6891-5p is expressed in B lymphocytes, supporting the hypothesis that miR-6891 plays a role in B lymphocyte homeostasis. Utilizing computational approaches, we have identified significantly enriched targets of miR-6891-5p and enriched pathways of significant, targeted genes. Methods In silico RNA folding, multiple sequence alignment and sequence logo plots of annotated IMGT HLA-B intron 4 sequence variants were generated using MATLAB to identify energetically favorable pre-miRNA hairpins and visualize allele diversity. Twelve independent miRNA target prediction algorithms were run in order to identify significant miR-6891-5p targets (p ⩽ 0.05). Functional enrichment of miR-6891-5p targets was performed using DAVID. Results There are eight unique sequence variants of HLA-B intron 4, derived from 194 IMGT HLA-B alleles with fully characterized intron 4 sequences. Every sequence variant is found to form stable pre-miRNA hairpin structures with permissible free-energies. MiR-6891-5p harbors no polymorphisms across annotated alleles, whereas miR-6891-3p was found to harbor two polymorphic positions, including one at position 5 of the seed region. Focused interpretation of high confidence, 3’ UTR targets of miR-6891-5p reveals enrichment of both T-cell and B-cell receptor signaling pathways. Conclusions Our analysis reveals that the HLA-B intron 4 derived miRNA, miR-6891-5p plays a role in regulating T-cell and B-cell receptor signaling pathways through translational suppression of targeted mRNA transcripts. Download : Download full-size image

A novel Transcript is Up‐Regulated by Fasting in the Hypothalamus and Enhances Insulin Signalling

A transcript of unknown function, regulated by fasting and feeding, was identified by microarray analysis. The transcript is up-regulated in the fasting state. An 1168-bp cDNA was cloned from rat hypothalamus and sequenced. This sequence is consistent with adipogenesis down-regulating transcript 3 (AGD3) (also known as human OCC-1) mRNA. A protein sequence identical to AGD3 was determined by mass spectrometry. In the rat brain, AGD3 mRNA is distributed in the arcuate nucleus, ventromedial hypothalamus, amygdaloid nuclei, hippocampus, and somatic cortex. Double in situ hybridisation showed that AGD3 mRNA is co-localised with pro-opiomelanocortin and neuropeptide Y in arcuate nucleus neurones. AGD3 binds with insulin receptor substrate 4 and increases insulin-stimulated phospho-Akt and regulates AMP-activated protein kinase and mammalian target of rapamycin downstream target S6 kinase phosphorylation.

Differential salivary gland transcript expression profile in Ixodes scapularis nymphs upon feeding or flavivirus infection

Ixodid ticks are vectors of human diseases such as Lyme disease, babesiosis, anaplasmosis, and tick-borne encephalitis. These diseases cause significant morbidity and mortality worldwide and are transmitted to humans during tick feeding. The tick–host–pathogen interface is a complex environment where host responses are modulated by the molecules in tick saliva to enable the acquisition of a blood meal. Disruption of host responses at the site of the tick bite may also provide an advantage for pathogens to survive and replicate. Thus, the molecules in tick saliva not only aid the tick in securing a nutrient-rich blood meal, but can also enhance the transmission and acquisition of pathogens. To investigate the effect of feeding and flavivirus infection on the salivary gland transcript expression profile in ticks, a first-generation microarray was developed using ESTs from a cDNA library derived from Ixodes scapularis salivary glands. When the salivary gland transcript profile in ticks feeding over the course of 3 days was compared to that in unfed ticks, a dramatic increase in transcripts related to metabolism was observed. Specifically, 578 transcripts were up-regulated compared to 151 down-regulated transcripts in response to feeding. When specific time points post attachment were analyzed, a temporal pattern of gene expression was observed. When Langat virus-infected ticks were compared to mock-infected ticks, transcript expression changes were observed at all 3 days of feeding. Differentially regulated transcripts include putative secreted proteins, lipocalins, Kunitz domain-containing proteins, anti-microbial peptides, and transcripts of unknown function. These studies identify salivary gland transcripts that are differentially regulated during feeding or in the context of flavivirus infection in Ixodes scapularis nymphs, a medically important disease vector. Further analysis of these transcripts may identify salivary factors that affect the transmission or replication of tick-borne flaviviruses.

Homozygous deletion of a gene-free region of 4p15 in a child with multiple anomalies: Could biallelic loss of conserved, non-coding elements lead to a phenotype?

We report a male patient, offspring of a consanguineous marriage between first cousins, with cognitive impairment, autistic-like behavior, deafness, postaxial polydactyly, and mild dysmorphic features. aCGH revealed a 600 kb homozygous deletion of 4p15.1 (from 33.553 to 34.159 Mb in NCBI36 hg18) encoding several transcripts of unknown function. Both parents are heterozygous for the deletion and the non-affected brother is homozygous for the normal alleles. We hypothesize that this deletion is likely to contribute to the phenotype of the patient. This case underlines the contribution of aCGH in discovering potentially pathogenic CNVs in consanguineous matings.

Identification and expression profiling of drought-regulated genes in mulberry (Morus sp.) by suppression subtractive hybridization of susceptible and tolerant cultivars

Mulberry, the backbone of sericulture industry, is a rainfed crop, and its biomass production is affected adversely under drought conditions. In this study, genes expressed differentially during drought stress response have been examined by PCR-Select subtractive hybridization. The sensitive and tolerant genotypes were identified based on physiological evaluation by determination of proline content, electrolyte leakage, and measurement of relative water content. In total, 1,920 clones were sequenced, representing 208 contigs and 151 singletons. The expressed sequence tags generated from this subtracted cDNA library comprises a broad repertoire of stress-responsive genes, which contribute to the process of drought tolerance in mulberry. Additionally, 23% of the cDNA library is represented by transcripts of unknown function. The expression of a select number of these drought-inducible genes was studied based on cDNA macroarray and Northern blot analyses. In order to unravel the crosstalk with other abiotic stresses, expression profile of Arabidopsis homologs of selected genes in response to a wide range of different stresses was studied using Genevestigator as a reference expression database. The results of this study show that subtractive hybridization coupled with validation steps for differential screening is an effective method for identification of stress/drought-induced genes in plants with limited sequence information available.

Somitovasculin, a Novel Endothelial-Specific Transcript Involved in the Vasculature Development

We recently isolated and characterized endothelial-like CD31(+) cells derived from mouse embryonic stem (mES) cells and identified their transcriptome. The main objective of this study was to determine the functional relevance of the transcripts of unknown function (TUF) for vasculature development. We selected 2 TUFs of more than 27 to study their role for blood vessel development in zebrafish. Morpholino (MO) knockdown of the zebrafish orthologs of the first TUF (TUF1, mouse cDNA BC022623) showed disruption of the intersegmental vessels (ISV) at 2 days postfertilization as observed by live imaging of fli:EGFP-transgenic embryos. The morphants showed abnormal blood circulation, but no effect on hematopoiesis was observed as demonstrated by gata-1 in situ hybridizations. Because knockdown of TUF1 resulted in disruption of the ISV patterning we named the TUF1 somitovasculin. TUF2 has been identified as cDNA clone BC020535. The MO knockdown of TUF2 resulted in a phenotype with an enlarged heart and the embryos lacked circulation completely. We have shown the participation of a novel transcript (named somitovasculin) in circulatory vessel development. The combination of expression profiling in differentiating mES cells and the zebrafish model has the potential for rapid identification and functional characterization of TUFs.

Transcripts of unknown function in multiple-signaling pathways involved in human stem cell differentiation

Mammalian transcriptome analysis has uncovered tens of thousands of novel transcripts of unknown function (TUFs). Classical and recent examples suggest that the majority of TUFs may underlie vital intracellular functions as non-coding RNAs because of their low coding potentials. However, only a portion of TUFs have been studied to date, and the functional significance of TUFs remains mostly uncharacterized. To increase the repertoire of functional TUFs, we screened for TUFs whose expression is controlled during differentiation of pluripotent human mesenchymal stem cells (hMSCs). The resulting six TUFs, named transcripts related to hMSC differentiation (TMDs), displayed distinct transcriptional kinetics during hMSC adipogenesis and/or osteogenesis. Structural and comparative genomic characterization suggested a wide variety of biologically active structures of these TMDs, including a long nuclear non-coding RNA, a microRNA host gene and a novel small protein gene. Moreover, the transcriptional response to established pathway activators indicated that most of these TMDs were transcriptionally regulated by each of the two key pathways for hMSC differentiation: the Wnt and protein kinase A (PKA) signaling pathways. The present study suggests that not only TMDs but also other human TUFs may in general participate in vital cellular functions with different molecular mechanisms.

Allele-Specific Gene Expression Is Widespread Across the Genome and Biological Processes

Allelic specific gene expression (ASGE) appears to be an important factor in human phenotypic variability and as a consequence, for the development of complex traits and diseases. In order to study ASGE across the human genome, we have performed a study in which genotyping was coupled with an analysis of ASGE by screening 11,500 SNPs using the Mapping 10 K Array to identify differential allelic expression. We found that from the 5,133 SNPs that were suitable for analysis (heterozygous in our sample and expressed in peripheral blood mononuclear cells), 2,934 (57%) SNPs had differential allelic expression. Such SNPs were equally distributed along human chromosomes and biological processes. We validated the presence or absence of ASGE in 18 out 20 SNPs (90%) randomly selected by real time PCR in 48 human subjects. In addition, we observed that SNPs close to -but not included in- segmental duplications had increased levels of ASGE. Finally, we found that transcripts of unknown function or non-coding RNAs, also display ASGE: from a total of 2,308 intronic SNPs, 1510 (65%) SNPs underwent differential allelic expression. In summary, ASGE is a widespread mechanism in the human genome whose regulation seems to be far more complex than expected.

Transcript expression profile of water-limited roots of hexaploid wheat (Triticum aestivum‘Opata’)

Triticum aestivum 'Opata' is an elite hard red spring wheat that has been used as a parent of the ITMI (International Triticeae Mapping Inititative) mapping population and also in the production of synthetically derived hexaploid wheats, some of which (following selection) show increased drought tolerance relative to Opata. Here, we describe the response of Opata roots to water withholding, using physiological variables and oligonucleotide microarrays. We identified 394 distinct transcripts whose abundance differed (p < or = 0.05) at least 1.5-fold between water-limited and control roots of Opata, of which 190 transcripts increased and 204 decreased following water limitation. In addition to previously characterized markers of abiotic stress and many genes of unknown function, we identified multiple putative glucanases and class III peroxidases as being particularly responsive to stress. We also compared these data to previously described microarray analyses of Opata's more drought-tolerant, synthetic-derived progeny, and found a relatively high correlation (r = 0.7) between responsive transcripts in the two genotypes, despite differing physiological responses. Some of the transcripts that we confirmed by qRT-PCR as being differentially expressed between Opata and the more tolerant synthetic-derived genotype under stress include a class III peroxidase, an AP2-family transcription factor, and several transcripts of unknown function.

Mapping the C. elegans noncoding transcriptome with a whole-genome tiling microarray

The number of annotated protein coding genes in the genome of Caenorhabditis elegans is similar to that of other animals, but the extent of its non-protein-coding transcriptome remains unknown. Expression profiling on whole-genome tiling microarrays applied to a mixed-stage C. elegans population verified the expression of 71% of all annotated exons. Only a small fraction (11%) of the polyadenylated transcription is non-annotated and appears to consist of approximately 3200 missed or alternative exons and 7800 small transcripts of unknown function (TUFs). Almost half (44%) of the detected transcriptional output is non-polyadenylated and probably not protein coding, and of this, 70% overlaps the boundaries of protein-coding genes in a complex manner. Specific analysis of small non-polyadenylated transcripts verified 97% of all annotated small ncRNAs and suggested that the transcriptome contains approximately 1200 small (<500 nt) unannotated noncoding loci. After combining overlapping transcripts, we estimate that at least 70% of the total C. elegans genome is transcribed.

Origin of phenotypes: Genes and transcripts

While the concept of a gene has been helpful in defining the relationship of a portion of a genome to a phenotype, this traditional term may not be as useful as it once was. Currently, "gene" has come to refer principally to a genomic region producing a polyadenylated mRNA that encodes a protein. However, the recent emergence of a large collection of unannotated transcripts with apparently little protein coding capacity, collectively called transcripts of unknown function (TUFs), has begun to blur the physical boundaries and genomic organization of genic regions with noncoding transcripts often overlapping protein-coding genes on the same (sense) and opposite strand (antisense). Moreover, they are often located in intergenic regions, making the genic portions of the human genome an interleaved network of both annotated polyadenylated and nonpolyadenylated transcripts, including splice variants with novel 5' ends extending hundreds of kilobases. This complex transcriptional organization and other recently observed features of genomes argue for the reconsideration of the term "gene" and suggests that transcripts may be used to define the operational unit of a genome.

Whole genome microarray analysis of gene expression in an imprinting center deletion mouse model of Prader–Willi syndrome

Prader-Willi syndrome (PWS) is caused by loss of paternally expressed genes in the 15q11-q13 region. To further characterize alterations in gene expression in this classical obesity syndrome we used whole genome microarrays to study a PWS mouse model resulting from a paternally derived imprinting center (IC) deletion (PWS IC deletion). These mice die generally within 2-3 days of life (reflective of failure to thrive in infants with PWS) and therefore, the analysis was performed on RNA extracted from the whole brain of PWS IC deletion mice and normal littermates at less than 24 hr after birth. Of more than 45,000 probes examined, 26,471 (59%) were detected for further analysis, and 69 had a significant change in expression of at least 1.5-fold and a false discovery rate (FDR) of 5%. Eight of the genes with differential expression were imprinted and from the PWS critical region (PWSCR). The three genes with the highest expression in the PWS IC mice were pro-opiomelanocortin (Pomc) and two transcripts of unknown function. Pomc knockout mice have been shown to develop obesity. Therefore, elevated Pomc RNA in PWS IC deletion neonatal mice may be an important genetic factor in the survival of these mice as it may affect eating behavior. Interestingly, Mc5r, a melanocortin receptor known to directly respond to Pomc expression changes, was upregulated as well. Mc5r is known to be involved with thermoregulation which is reportedly abnormal in PWS infants. These observations support a role for Pomc and the network of genes involved in regulating energy homeostasis in the early clinical findings of failure to thrive observed in PWS. Other notable patterns include three previously unstudied transcripts that are expressed only from the paternal allele under regulatory control of the IC and include AK013560, BB3144814, and BB182944 (whose genes are located in the mouse PWSCR on chromosome 7B). As expected, all the known paternally expressed genes from the PWSCR had detection signals below the threshold in the PWS IC deletion mice but were clearly detectable in control littermates. Several of the genes in this study were further examined by quantitative reverse transcription-PCR (RT-PCR) to confirm their expression status. Further analysis of gene expression in these mice may lead to novel pathways affected in PWS. These results, along with other recent reports, suggest that the cumulative effect of modest changes in expression of many genes, especially genes involved in energy metabolism, contribute to the failure to thrive of infants with PWS.

Over- and Underdosage of SOX3 Is Associated with Infundibular Hypoplasia and Hypopituitarism

Duplications of Xq26-27 have been implicated in the etiology of X-linked hypopituitarism associated with mental retardation (MR). Additionally, an expansion of a polyalanine tract (by 11 alanines) within the transcription factor SOX3 (Xq27.1) has been reported in patients with growth hormone deficiency and variable learning difficulties. We report a submicroscopic duplication of Xq27.1, the smallest reported to date (685.6 kb), in two siblings with variable hypopituitarism, callosal abnormalities, anterior pituitary hypoplasia (APH), an ectopic posterior pituitary (EPP), and an absent infundibulum. This duplication contains SOX3 and sequences corresponding to two transcripts of unknown function; only Sox3 is expressed in the infundibulum in mice. Next, we identified a novel seven-alanine expansion within a polyalanine tract in SOX3 in a family with panhypopituitarism in three male siblings with an absent infundibulum, severe APH, and EPP. This mutation led to reduced transcriptional activity, with impaired nuclear localization of the mutant protein. We also identified a novel polymorphism (A43T) in SOX3 in another child with hypopituitarism. In contrast to findings in previous studies, there was no evidence of MR or learning difficulties in our patients. We conclude that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily MR.

Endurance training-induced changes in insulin sensitivity and gene expression

The beneficial effects of regular physical activity on insulin sensitivity (SI) and glucose tolerance are well documented, with considerable heterogeneity in responsiveness to exercise training (ET). To find novel candidate genes for ET-induced improvement in SI, we used microarray technology. Total RNA was isolated from vastus lateralis muscle before and after 20 wk of exercise from individuals participating in the HERITAGE Family Study. SI index was derived from a frequently sampled intravenous glucose tolerance test using MINMOD Millennium software. Sixteen subjects were selected: eight showing no changes in SI (low responders, LSIR) and eight displaying marked improvement in SI (high responders, HSIR) with ET. The SI increase was about four times greater in HSIR compared with LSIR (+3.6 +/- 0.5 vs. -1.2 +/- 0.5 microU.ml(-1).min(-1), mean +/- SE), whereas age, body mass index, percent body fat, and baseline SI were similar between the groups. Triplicate microarrays were performed, comparing pooled RNA with HSIR and LSIR individuals for differences in gene expression before and after ET using in situ-generated microarrays (18, 861 genes). Array data were validated by quantitative RT-PCR. Almost twice as many genes showed at least twofold differences between HSIR and LSIR after training compared with pretraining. We identified differentially expressed genes involved in energy metabolism and signaling, novel structural genes, and transcripts of unknown function. Genes of interest upregulated in HSIR include V-Ski oncogene, four-and-a-half LIM domain 1, and titin. Further study of these novel candidate genes should provide a better understanding of molecular mechanisms involved in the improvement in insulin sensitivity in response to regular exercise.

Differential gene expression in auristatin PHE-treated Cryptococcus neoformans.

The antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen Cryptococcus neoformans. To gain a broader understanding of the cellular response of C. neoformans to auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 micro M) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example, RHO1 and an open reading frame (ORF) encoding a protein with 91% similarity to the Schizophyllum commune 14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in Pleurotus sajor-caju was up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for C. neoformans. This is the first report of drug action using DD in C. neoformans.

A Physical and Transcriptional Map of the Preaxial Polydactyly Locus on Chromosome 7q36

Preaxial polydactyly is a congenital hand malformation that includes duplicated thumbs, various forms of triphalangeal thumbs, and duplications of the index finger. A locus for preaxial polydactyly has been mapped to a region of 1.9 cM on chromosome 7q36 between polymorphic markers D7S550 and D7S2423. We constructed a detailed physical map of the preaxial polydactyly candidate region. With a combination of methods we identified and positioned 11 transcripts within this map. By recombination analysis on families with preaxial polydactyly, using newly developed polymorphic markers, we were able to reduce the candidate region to approximately 450 kb. The homeobox geneHLXB9,a putative receptor C7orf2, and two transcripts of unknown function, C7orf3 and C7orf4, map in the refined candidate region and have been subjected to mutation analysis in individuals with preaxial polydactyly.

Preliminary characterization of the protein encoded by human testis-enhanced gene transcript (TEGT).

TEGT is a conserved, widely expressed gene transcript of unknown function that has been studied previously only at the nucleic acid level. The deduced amino acid sequence predicts a highly hydrophobic, 26.5 kDa integral membrane protein with seven potential transmembrane domains. Little else is known about TEGT protein because of the lack of definitive homology to other known sequences and the absence of informative consensus motifs. The present report details a preliminary study of human TEGT (hTEGT) protein. (i) In vitro translation of hTEGT in reticulocyte lysates required the presence of microsomes for efficient synthesis, suggesting that hTEGT must target to the endoplasmic reticulum to be translated. Immunofluorescence of cells transiently expressing haemagglutinin-tagged hTEGT localized the protein mainly to the endoplasmic reticulum. The protein demonstrated no obvious post-translational modifications such as signal-peptide cleavage, N-linked glycosylation or O-linked glycosylation. (ii) Both hTEGT and haemagglutinin-tagged hTEGT appeared to retain partial secondary and tertiary structure in the presence of SDS. Both electrophoresed as a broad band or doublet with apparent molecular weights of 22-24.5 kDa on SDS-PAGE, aggregated either homotypically or heterotypically when boiled in SDS, and were toxic after 24 h when highly overexpressed in 293 T cells. These properties are believed to be caused by the protein's hydrophobicity. (iii) The protein appeared to associate strongly with other intracellular molecules since haemagglutinin-tagged hTEGT was extracted poorly from transiently transfected HeLa cells. Further study will be required to determine the cellular function of TEGT.