Comparison of Telomere Length between Buccal Cells and Blood Cells.

Telomere length (TL) in blood cells is commonly used as a proxy for TL in other tissue types. The source of DNA of adequate quality and quantity is important for TL analysis. Compared to blood cells, buccal cells easy for genomic DNA preparation would facilitate the rapid and reliable TL analysis. However, the feasibility of buccal cells for TL analysis remains yet unestablished. We characterized TL of buccal cells and blood cells collected from 52 individuals using buccal cell swabs and fingertip sticks. Relative TL (RTL) determined by quantitative PCR showed that there is a strong correlation between buccal RTL and blood RTL (r=0.877, p<0.001), suggesting that buccal cells are adequate sources of DNA for TL analysis. The validity of sampling using buccal cell swabs provides simple operation and good reproducibility for TL analysis, that overcomes the discomfort and risk of infection caused by blood sampling.

Quantitative determination of endogenous cardiolipin in rat cerebral tissues by high-performance liquid chromatography using standard addition method.

Cardiolipin is an important cellular component, and its normal level is a key prerequisite for maintaining the structure and function of mitochondria. To accurately quantify endogenous cardiolipin content in mitochondria, a standard addition method (SAM) was developed to establish a high-performance liquid chromatographic (HPLC) technique that is both reliable and accurate. Increasing amounts of cardiolipin standards were added to a constant amount of isolated mitochondria prior to the extraction procedure, and the two were extracted together. By limiting the interference effects that occur to within an acceptable range in an analytical system examined, this procedure ensures an ideal match of the sample composition in the standards, even if the composition is extremely complex or completely unknown. Then, the desired results can be obtained by extrapolation. As such, the authentic content of the endogenous cardiolipin can be obtained with greater accuracy than with classical detection methods, e.g. external standard calibration (ESC) and internal standard calibration (ISC). This method provides an excellent means of quantifying endogenous substances in living cells. The authors expect this method to be useful for researchers working on mitochondria-related mechanisms, cell survival-related mechanisms and similar topics.

Cav-1 deletion impaired hematopoietic stem cell function.

A tightly controlled balance between hematopoietic stem and progenitor cell compartments is required to maintain normal blood cell homeostasis throughout life, and this balance is regulated by intrinsic and extrinsic cellular factors. Cav-1 is a 22-kDa protein that is located in plasma membrane invaginations and is implicated in regulating neural stem cell and embryonic stem cell proliferation. However, the role of Cav-1 in hematopoietic stem cell (HSC) function is largely unknown. In this study, we used Cav-1(-/-) mice to investigate the role of Cav-1 in HSCs function during aging. The results showed that Cav-1(-/-) mice displayed a decreased percentage of B cells and an increased percentage of M cells in the bone marrow and peripheral blood, and these changes were due to an increased number of HSCs. FACS analysis showed that the numbers of Lin(-)Sca1(+)c-kit(+) cells (LSKs), long-term HSCs (LT-HSCs), short-term HSCs and multipotent progenitors were increased in Cav-1(-/-) mice compared with Cav-1(+/+) mice, and this increase became more pronounced with aging. An in vitro clonogenic assay showed that LT-HSCs from Cav-1(-/-) mice had reduced ability to self-renew. Consistently, an in vivo competitive transplantation assay showed that Cav-1(-/-) mice failed to reconstitute hematopoiesis. Moreover, a Cav-1 deletion disrupted the quiescence of LSKs and promoted cell cycle progression through G2/M phase. In addition, we found that Cav-1 deletion impaired the ability of HSCs to differentiate into mature blood cells. Taken together, these data suggest that Cav-1-deficient cells impaired HSCs quiescence and induced environmental alterations, which limited HSCs self-renewal and function.

Herpes simplex virus type-1 infection upregulates cellular promoters and telomerase activity in both tumor and nontumor human cells.

Targeted gene expression through viral vectors has been a promising approach for gene therapy. However, the effects of viral gene products expressed from virus vectors on the expression of the host gene are not well known. In the present study, we examined the activities of cellular promoters, including the promoter for genes of human telomerase reverse transcriptase (hTERT), tyrosinase and probasin, in both tumor and normal cells after infection with herpes simplex virus type 1 (HSV-1) vectors. Our results showed that infection with replication-defective HSV-1 vectors significantly upregulated the activity of all three cellular promoters in a nonsequence specific fashion in all cell types tested. Furthermore, viral infection upregulated activities of the hTERT promoter and endogenous telomerase in nontumoral cells. Additional experiments suggested that the viral immediate-early gene product, infected cell protein 0, might be responsible for the deregulation of cellular promoter activity and activation of telomerase. Our study alerts to the potential risk of oncogenesis through deregulation of host gene expression, such as the telomerase by viral vectors in normal cells.

Histone deacetylation is involved in the transcriptional repression of hTERT in normal human cells.

Trancriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic protein of human telomerase, plays a critical role in the activation of the enzyme during cell immortalization and tumorigenesis. However, the molecular mechanisms involved in the regulation of hTERT expression are still not fully understood. We have previously cloned and characterized the genomic sequences and promoter of the hTERT gene. Here, we provide evidence that histone deacetylation is involved in the repression of hTERT in human cells. Inhibition of histone deacetylases by trichostatin A in telomerase-negative cells resulted in activation of telomerase activity and up-regulation of hTERT mRNA. Transient transfection experiments with a reporter under control of the hTERT promoter indicated that this promoter can be activated by trichostatin A. Finally, our results show that repression of the hTERT promoter by the Mad protein requires histone deacetylase activity, whereas de-repression by trichostatin A is independent of the E-boxes located in its core region.

Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells.

In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5'-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor alpha (ERalpha). In vivo DNA footprinting revealed specific modifications of the ERE region in ERalpha-positive but not ERalpha-negative cells upon treatment with 17beta-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERalpha but not ERbeta remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter.

Telomerase, the enzyme that synthesizes telomeric DNA, is not expressed in most human somatic cells but is activated with in vitro immortalization and during tumorigenesis, and repressed by cell differentiation. Of the two components of the core enzyme, the catalytic protein hTERT is limiting for activity. To investigate mechanisms of hTERT gene regulation, we have cloned genomic sequences encompassing the complete hTERT transcription unit. The hTERT gene consists of 16 exons and 15 introns spanning approximately 35 kb. Transient transfections of immortal human cells with potential regulatory 5' sequences linked to a reporter, combined with deletion analysis of these sequences, indicated that elements responsible for promoter activity are contained within a region extending from 330 bp upstream of the ATG to the second exon of the gene. Assays in different cell types have shown that the hTERT promoter is inactive in normal and in transformed pre-immortal cells, but, like telomerase, it is activated with cell immortalization. Sequence analysis revealed that the hTERT promoter is GC-rich, lacks TATA and CAAT boxes but contains binding sites for several transcription factors that may be involved in its regulation. The abundance of these sites suggests the possibility that hTERT expression may be subject to multiple levels of control and be regulated by different factors in different cellular contexts.

Transactivation by expression of the hepatitis B virus X protein with an inducible system.

We describe here the conditional expression of the hepatitis B virus X protein using the inducible system controlled by a tet-responsive promoter. Induction of the X protein in Rat-2 fibroblasts activated transcription from a heterologous gene promoter and stimulated the DNA-binding activity of NFkB. The ability to produce this biologically active X protein in a stable cell line will accelerate the elucidation of the function and mechanisms of X and eventually help us understand the role of X in natural viral infection and carcinogenesis.

Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human cells.

Telomere shortening in human somatic cells and telomere maintenance in most human immortal cell lines and tumours correlate respectively with the absence and presence of telomerase, the enzyme that synthesizes telomeric DNA de novo . However, approximately 30% of in vitro immortalized human cell lines do not express this enzyme and maintain telomeres by an alternative pathway (ALT) that may also operate in some tumours. Human telomerase is a reverse transcriptase comprising minimally an RNA subunit (hTER) and a catalytic protein moiety (hTERT). Normal somatic cells retain expression of hTER but not of hTERT, and can be converted to a telomerase-positive phenotype by ectopic expression of the catalytic protein. We similarly have restored enzymatic activity to those ALT cell lines that retain hTER expression. We also report that in those ALT cells that are hTER negative, reintroduction of both hTER and hTERT is necessary and sufficient for conversion to telomerase positivity. Moreover, transfection of these cells with hTERT in conjunction with hTERs with a mutated template results in the expression of an enzyme with altered specificity. Reconstitution of telomerase activity in ALT cells, particularly an activity capable of synthesizing mutant telomeric DNA, may be exploited for the study of the ALT mechanism and its interaction with the telomerase-dependent pathway, and for assessing the effects of mutant telomeres on cell viability.

Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs.

Active transport of proteins into the nucleus is mediated by interaction between the classical nuclear localization signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex. This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival. Using a fragment of BRCA1 containing the two NLS motifs as a bait for yeast two-hybrid screening, we have isolated four clones, one of which is importin alpha. Here we characterize one of the other clones identified, BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in human mammary epithelial cells and some but not all tissues of mice. The isolated full-length cDNA encodes a novel protein containing 600 amino acid residues with pI 6.04. Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats are present in the middle and C-terminal regions of the protein, respectively. BRAP2 also shares significant homology with a hypothetical protein from yeast Saccharomyces cerevisiae, especially in the zinc finger region. Antibodies prepared against the C-terminal region of BRAP2 fused to glutathione S-transferase specifically recognize a cellular protein with a molecular size of 68 kDa, consistent with the size of the in vitro translated protein. Cellular BRAP2 is mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar specificity to that of importin alpha in both two-hybrid assays in yeast and glutathione S-transferase pull-down assays in vitro. Other motifs such as the SV40 large T antigen NLS motif and the bipartite NLS motif found in mitosin are also recognized by BRAP2. Similarly, the yeast homolog of BRAP2 also binds to these NLS motifs in vitro. These results imply that BRAP2 may function as a cytoplasmic retention protein and play a role in regulating transport of nuclear proteins.

The hepatitis B virus X-associated protein, XAP3, is a protein kinase C-binding protein.

The hepatitis B virus X protein induces transcriptional activation of a wide variety of viral and cellular genes. In addition to its ability to interact directly with many nuclear transcription factors, several reports indicate that the X protein stimulates different cytoplasmic kinase signal cascades. Using the yeast two-hybrid screen, we have isolated a clone designated X-associated protein 3 (XAP3) that encodes a human homolog of the rat protein kinase C-binding protein. One of the activation domains of X (amino acids 90-122) is required for binding to XAP3, while the NH2-terminal part of XAP3 is necessary for binding to X. Both X and XAP3 bound specifically to the eta PKC isoenzyme synthesized in rabbit reticulocyte lysates. Overexpression of XAP3 enhanced X transactivation activity. These results support earlier findings that one of the mechanisms of transactivation by X is through involvement with the cellular protein kinase C pathway.

XAP2, a novel hepatitis B virus X-associated protein that inhibits X transactivation.

The hepatitis B virus X protein is a promiscuous transcriptional transactivator. Transactivation by the X protein is most likely mediated through binding to different cellular factors. Using the yeast two-hybrid method, we have isolated a clone that encodes a novel X-associated cellular protein: XAP2. X and XAP2 interactions also occur in vitro. Antiserum raised against XAP2 recognizes a cytoplasmic protein with an apparent molecular mass of 36 kDa. The interaction between X and XAP2 requires a small region on X containing amino acids 13-26. From Northern blot analyses, XAP2 is ubiquitously expressed in both liver-derived and non-liver-derived cell lines as well as in normal non-liver tissues. In contrast, XAP2 is expressed in very low level in the normal human liver. In transfection assays, overexpression of XAP2 abolishes transactivation by the X protein. Based on these results, we suggest that XAP2 is an important cellular negative regulator of the X protein, and that X-XAP2 interaction may play a role in HBV pathology.

Seripauperins of Saccharomyces cerevisiae: a new multigene family encoding serine-poor relatives of serine-rich proteins.

A gene, PAU1, has been cloned from Saccharomyces cerevisiae and sequenced. It is located in a telomeric region, probably on chromosome IV, and contains an open reading frame encoding a protein of 120 amino acids (aa) (approx. 13 kDa). The deduced sequence is nearly identical to two other genes found in GenBank (named PAU2 and PAU3 by us), which are located close to the ends of chromosomes V and III, respectively. Blotting of separated chromosomes with a PAU1 probe at high stringency revealed that at least six chromosomes in addition to III, IV and V possessed related sequences, suggesting a large gene family. Probing of an ordered array of phage lambda clones containing yeast genomic DNA inserts ('Olson filters') revealed ten additional hybridizing sequences, located close to the ends of the left and/or right arms of chromosomes I, II, VII, VIII, X, XII, XIV and XV. Transcription of these sequences could not be demonstrated, however, under a wide variety of growth and culture conditions. The deduced PAU1, PAU2 and PAU3 aa sequences are all highly homologous with the SRP1 aa sequences, which contains eight serine-rich tandem repeats of 12 aa each, at its C terminus. This homology is limited, however, to the N-terminal half of SRP1, and does not include the repeats. In fact, PAU1 is quite serine-poor (5.8%), leading to the suggested name of seripauperins for this family of genes.

Creation of a functional promoter by rearrangement in a Kluyveromyces lactis linear plasmid.

The expression of genes from cytoplasmic killer plasmids in the yeast Kluyveromyces lactis depends on their own specific transcription system. Therefore, the kanamycin/G418-resistance-encoding gene, KmR, under its natural promoter cannot be expressed when integrated into the pGKL1 plasmid. However, one G418R transformant clone was isolated. The resistance was due to the presence of two modified plasmids, k1-kan2a (10.4 kb) and k1-kan2b (5.2 kb) which were derivatives of pGKL1 containing the KmR gene. In these mutant plasmids, a large part of pGKL1 has been replaced by the KmR gene harboring a rearranged 5'-flanking region extending over 600 bp. This new DNA sequence has been cloned and sequenced. The rearranged sequence allowed the KmR gene to be expressed at high level, enabling the transformant cells to grow on a medium containing G418 at 2 mg/ml. This high level of resistance was found to be due to increased transcription of the KmR gene. Primer extension experiments suggested that the rearranged upstream region of KmR contained transcription promoting sites recognized by the killer-plasmid-specific transcription system.

Linear DNA plasmids from Pichia etchellsii, Debaryomyces hansenii and Wingea robertsiae.

Linear DNA plasmids were found in the following yeasts: four strains of Kluyveromyces lactis, one of Debaryomyces hansenii, one of Wingea robertsiae and four of Pichia etchellsii. In each case, the plasmids were present as a pair of DNA molecules of different sizes. The plasmids of K. lactis strains were associated with a killer activity and their structure was similar to the known killer plasmids pGKL1 and 2. The plasmids from the other three species were different from pGKL plasmids and showed no killer activity against the yeast species tested so far. In all cases, the linear molecules possessed terminal (probably inverted) repeats and their 5' ends had a protected structure insensitive to lambda exonuclease, while the 3' ends were accessible to exonuclease III. All these strains could be efficiently cured of the plasmids by ultraviolet irradiation. The plasmids from D. hansenii (pDH1A and B) and from W. robertsiae (pWR1A and B) shared related sequences with some of the K. lactis killer plasmid genes (encoding the supposed DNA polymerases, RNA polymerase and the chitinase), suggesting related genome organization of these plasmids. The pair of plasmids from P. etchellsii (pPE1A and B) appear to be a distantly related member of the group. This pair showed no sequence homology with other plasmids, except weak homology with the putative RNA polymerase gene of pGKL2. None of the plasmids contained the sequences homologous to ORF3 and ORF4 of pGKL1 encoding the toxin resistance determinant and the toxin gamma subunit, respectively.

Characterization of a circular plasmid from the yeast Kluyveromyces waltii.

A new plasmid was found in the yeast Kluyveromyces waltii. This high-copy-number plasmid, named pKW1, is a double-stranded circular DNA plasmid of 5619 bp. It has several features characteristic of the 2 mu-type plasmids: presence of two inverted repeats and four open reading frames, as well as the interconversion of two isomeric forms. However, the nucleotide sequence shows little homology with known yeast plasmids. An ARS function was localized within a segment of 545 bp near one of the inverted repeats. Chimeric plasmids carrying this segment efficiently transformed K. waltii. A strain of K. waltii cured of the plasmid (cir degree) was also obtained. In the pKW1 sequence, a functionally neutral region was found at which foreign DNA can be inserted with little effect on plasmid stability. Such constructions carrying the full sequence of pKW1 replicated autonomously in a cir degree host and were particularly stable. pKW1-derived full-sequence plasmids also transformed K. thermotolerans, but not K. lactis.