No / The DNA content of hair [1.] and [2.] is typically low compared to other tissues, as hair cells undergo dehydration and catabolic breakdown of nucleic acids and organelles during keratinisation [3]. As a consequence, ancient hair specimens have not been widely used as a source of ancient DNA. However, mitochondrial DNA (mtDNA) has been extracted from degraded and old hair samples, including burnt specimens [4], 100-year-old Native American samples [5], and wool from a 9,400 year old Bighorn sheep [6]. We have investigated the potential of hair as an aDNA source by analyzing DNA survival in 12 samples which range from 60 to >64,800 years of age and their susceptibility to contamination with modern DNA. mtDNA was successfully amplified, cloned, and sequenced from 10 of the 12 hair samples following decontamination procedures (Table 1). DNA was quantified using Quantitative Real-Time PCR in a subset of the samples (Table 1). The survival of high copy numbers of 16S DNA from the 3,000 year-old Pazyryk horse hairs is consistent with the observation that DNA survives longer at sub-zero temperatures [7]. Of greater surprise was the persistence of high numbers of 16S and Control Region DNA molecules in hairs sampled from a bison mummy 14C dated to >64,800 years. This result was independently replicated and extends the time frame from which authentic DNA has been retrieved from hair by at least seven-fold, placing it on a par with the oldest authentic DNA retrieved from bones and teeth [8]. No nuclear DNA could be amplified from the bison hair, consistent with observations of modern hair samples [1.] and 9. M.R. Wilson, D. Polanskey, J. Butler, J.A. DiZinno, J. Replogle and B. Budowle, Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts, Biotechniques 18 (1995), pp. 662¿669.[9.]. It is probably significant that the bison hairs are exceedingly well preserved ¿ the atomic carbon to nitrogen ratio (3.47) is similar to modern mammal hair [10.] and [11.] and histological analysis of the specimen demonstrates the only structural modifications to be slight cuticular loss and adherent deposits (Supplemental data).

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Helix hand inversion exhibited by Bacillus subtilis macrofibers is induced by changes in culture medium composition. The kinetics of this inversion are compared to those of temperature-induced inversions. D-alanine evokes a similar inversion process. The role of left-twist proteins(s), the existence of "memory", and the asymmetry of left to right versus right to left kinetics are confirmed within the context of these inversion regimes. Initiation time of right to left inversions is correlated to degree of pre-shift twist. Evidence is presented suggesting effective twist of the wall is defined by (1) the average of that twist conformation inserted prior to a shift in culture conditions and that of wall inserted following the shift and (2) the location of left-handed material within the wall. A constant 50 minute delay is observed before initiation of left to right inversions, irregardless of twist. Evidence is presented for a protein in the left to right inversion process. A classification system of macrofiber phenotypes based upon hand and degree of structural organization has been established. Three major classes are identified. Subclasses are shown to be distinguishable. Isotwist phenotypes of seven strains are defined upon a matrix of temperature and medium composition. These plots reveal a fundamental pattern of hand and organization that is present in each of the strains studied. The polarity of the four axes, the range of attainable twist conformations, and the existence of a right-hand maximum in the 12.5% SPl domain remain virtually constant. Major variations include extent of a disorganized band and/or the shifting of conformational range either left or right. Several mutants were transformed into A734, a strain that produces the tightest structures at all four matrix corners. Multiple mutations are responsible for the phenotypes of several strains. Evidence is presented for single genes that express as extreme left-handedness and stress at high temperature, swelling and stress in TB at high temperature, and reduction in structural organization produced in high TB content at low temperature.

A small lipid-containing bacteriophage PRD1 specifies its own DNA polymerase which utilizes terminal protein as a primer for DNA synthesis. The PRD1 DNA polymerase gene has been sequenced and its amino acid sequence deduced. This protein-primed DNA polymerase consists of 553 amino acid residues with a calculated molecular weight of 63,300. Thus, it is the smallest DNA polymerase ever isolated from prokaryotic cells. Comparison of the PRD1 DNA polymerase with other DNA polymerases whose sequences have been published, yielded segmental but significant homologies. These results strongly suggest that many prokaryotic and eukaryotic DNA polymerase genes regardless of size have evolved from a common ancestral gene. The results further indicate that those DNA polymerases which use either an RNA or protein primer are related. We propose to classify DNA polymerases on the basis of their evolutionary relatedness. In order to overexpress PRD1 DNA polymerase in E. coli cells, the 2kb Hae II fragment was isolated from phage genomic DNA. This fragment was then cloned into pEMBLex3 expression vector. Phagemid pEMBLex3 contains lambda pR promoter and cI857 gene as a repressor. A specific 57 bp deletion was performed by using uracil containing ss DNA and oligonucleotide spanning each region to remove an unwanted non-coding region. After this deletion, the PRD1 DNA polymerase gene is totally under the control of the vector promoter and SD sequence. Upon heat induction, a protein with an apparent size of 68 kdal was overexpressed as an active PRD1 DNA polymerase. The expression of DNA polymerase was about 1% of total E. coli protein. The PRD1 DNA polymerase is a small multifunctional DNA polymerase and has three major conserved amino acid sequences which are shared among many DNA polymerases including human DNA polymerase alpha. Therefore, the PRD1 DNA polymerase provides an useful model system to study structure-function analysis of DNA polymerases. Four specific amino acid changes generated in conserved regions by the site-directed mutagenesis, in order to investigate their functional roles. Based on complementation test, three conserved regions are functional domains of PRD1 DNA polymerase.

Hypervariable minisatellite regions of human DNA are of considerable interest, not only as highly informative genetic systems, but also as intermediately sized regions of tandem repetition. Methods for the isolation of minisatellite loci from the human genome have been investigated, and 23 new hypervariable loci cloned from an ordered array Charomid library. This method not only allows very efficient isolation of human minisatellites, but can also be used to observe the degree of overlap between multi-locus DNA fingerprinting probes. The 23 new loci have a mean heterozygosity level of 71%, and have been characterized and mapped in the genome. The genomic disposition of human minisatellites has been analysed by investigation of cloned examples. The minisatellites studied show a strong tendency to cluster near the ends of chromosomes, and sequence analysis demonstrates a significant excess of dispersed repeat elements in the DNA flanking human minisatellites. Minisatellite variant repeat (MVR) mapping has also been used to investigate the internal structure of minisatellite alleles. Somatic allele length mutation events have been demonstrated in DNA from colorectal adenocarcinomas, and the mutations observed show many features of general similarity to germline mutation events. A series of human breast tumours has been screened for somatic change, using both multi-locus DNA fingerprinting probes and single-locus minisatellite probes. Somatic change in breast cancers is much less frequent than in colorectal tumours, but some allele losses and mutations have been defined, including a highly unusual mutation, which may be the result of a minisatellite transposition event. Finally, evolution at minisatellite loci has been studied, both by examination of allelic states in current human populations, as well as comparison with non-human primates.

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The role of the tumor suppressors p53 and p16 genes in the cellular response to Ultraviolet light. SUMMARY Proliferating cells respond to DNA damage by concomitantly arresting cellular growth at checkpointsa nd activating DNA repair processesC. ell cycle arrestsa re mediated,i n part, by the inhibition of cyclin-dependent kinases (CDKs), whose function is required for cell cycle progression. p53, p21WAF' and p16`NK4aa re the products of tumor suppressor genes that play important roles during the cellular response to genotoxic stresses. p53 and p16 coding genes have been found mutated or transcriptionally silenced in different cancer types both sporadic and familial. Indeed, p16 has been found to be linked to familial melanoma, whose etiology is related to sun-light induced DNA damage. It is hence important to ascertain whether p53 and p16 are involved in the cellular response to UV damage. In this report I present evidence that p53 is not involved in UV-induced cellular growth arrest in late G1 phase. This has been demonstrated in HeLa cells synchronized at the G1/S border by aphidicolin, followed by UV exposure. Interestingly, the length of this p53-independent G1 arrest has been shown to be UV dose-dependent. Similar results were also obtained with other p53-deficient cell lines, including the human promyelocytic leukemia HL-60 and mouse p53 knock-out cells. As expected, all of these cell lines were defective in v-ray-induced cell growth arrest at late G1Using different assays I also show here that p16-compromised U20S osteosarcoma cells are deficient in the removal of UV damage, as compared to their isogenic derivatives EH1 and EH2 counterparts that express p16. This deficiency is associated with a high level of UV-induced apoptosis, which is significantly reduced in the p16-expressing EH I, EH2 and p16+/+ mouse embryonic fibroblast (MEF) cells, indicating that p16 protects cells from undergoing apoptosis in response to UV light. Importantly, this reduction in UV-mediated apoptosis was associated with down-regulation of the pro-apoptotic Bax protein, with no effect on Bcl-2 expression, suggesting that this anti-apoptotic role of p16 is mediated via the intrinsic p53-dependent mitochondrial pathway. On the other hand, p16 sensitized cells to cisplatin-mediated apoptosis through Bcl-2 decline. Furthermore, I show that p16 is involved in UV-related G1 checkpoint and controls the expression and UV-dependent activation of another CDK inhibitor, p21wAFI. Importantly, this relationship between p16 and p21 exists also in MEFs, suggesting that it is not cell type- or species-dependent. These results indicate that, in addition to its role in cell cycle control and senescence, p16 also plays an important role in the cellular response to UV damage, possibly by inhibiting apoptosis and facilitating cell cycle arrest and photolesion removal. The data presented here provide further insights into the role of p53 and p16, as tumor suppressors, in carcinogenesis and have potential implications for future therapy strategies

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