Touchdown PCR Research Articles

Cloning and Analysis of the Promoter Region of Rat uPA Gene

Objective To clone and analyze the promoter sequence of rat urokinase plasminogen activator protein gene. Methods The genomic DNA was extracted from rat testicular tissue. According to urokinase plasminogen activator, the gene sense primer and antisense primer of uPA gene were designed and synthesized, then Touch-Down PCR were performed. After proper purification, the PCR product was sequenced, analyzed with the promoter prediction software and compared with the DNA sequence of rattuas urokinase plasminogen activator. Results The cloned uPA gene was about 1 572 bp in length, which contained a full open-reading frame with 21 bp in length exons, and the upper region of transcriptional start was 1 551 bp in length which was eucaryon transcriptional control area. The 5′ UTR had a promoter region including a non-responsive TATA-box. Not only the GC-box binding region was found in this gene, but also active protein 1(AP1) and SP1 were seen in other regions. Conclusion A 1 572 bp uPA gene fragment (GenBank accession No.X65651) was obtained from rat genomic DNA library, containing eucaryon transcriptional control area with a promoter region, non-conspicuous TATA-box, GC-box and an extron. A non-responsive TATA-box is located at the upper −30 region.

R2202 Touchdown PCR as a tool for improved detection of invasive candidosis

R2202 Touchdown PCR as a tool for improved detection of invasive candidosis

Expression of splice variants of 1α-hydroxylase in mcf-7 breast cancer cells

1,25-Dihydroxyvitamin D3 (calcitriol) is the most active natural metabolite of Vitamin D3. It has strong antiproliferative and differentiating effects on various cell types including breast cancer cells. 25-Hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase, CYP27B1) is one of the key enzymes in the formation of calcitriol. It has been found in breast cancer cells suggesting an autocrine regulation of formation of calcitriol in these cells. Alternative splicing of the encoding genes for this enzyme can possibly play a role in regulating the enzyme level and can explain tissue specific variations of 1α-hydroxylase activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. In order to obtain more information on the abundance of 1α-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The full-length sequence of 1α-hydroxylase and two different splice variants of this enzyme containing intron 1 were isolated. By Western blot technique we then confirmed the protein products of the full-length enzyme and its splice variants. We hypothesize that that the expression of splice variants can lead to a quantitatively lower expression of the mRNA of the full-length enzyme. The abundance of less active 1α-hydroxylase protein variants can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues.

Calmodulin-like Protein Increases Filopodia-dependent Cell Motility via Up-regulation of Myosin-10

Human calmodulin-like protein (CLP) is an epithelial-specific protein that is expressed during cell differentiation but down-regulated in primary cancers and transformed cell lines. Using stably transfected and inducible HeLa cell lines, we found that CLP expression did not alter the proliferation rate and colony-forming potential of these cells. However, remarkable phenotypic changes were observed in CLP-expressing compared with control cells. Soft agar colonies of CLP-expressing cells had rough boundaries, with peripheral cells migrating away from the colony. Cells expressing CLP displayed a striking increase in the number and length of myosin-10-positive filopodia and showed increased mobility in a wound healing assay. This increase in wound healing capacity was prevented by small interference RNA-mediated down-regulation of myosin-10. Fluorescence microscopy and Western blotting revealed that CLP expression results in up-regulation of its target protein, myosin-10. This up-regulation occurs at the protein level by stabilization of myosin-10. Thus, CLP functions by increasing the stability of myosin-10, leading to enhanced myosin-10 function and a subsequent increase in filopodial dynamics and cell migration. In stratified epithelia, CLP may be required during terminal differentiation to increase myosin-10 function as cells migrate toward the upper layers and establish new adhesive contacts.

Cloning and expression of gene encoding the thermostable direct hemolysin from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis of crimson snapper (Lutjanus erythopterus)

The main aims of this study were to clone and express complete open reading frame (ORF) of thermostable direct haemolysin gene (tdh) from Vibrio alginolyticus strain HY9901 in Escherichia coli, and further evaluate the virulence of expressed TDH on mouse and crimson snapper. A 410 bp internal fragment of the tdh gene was amplified by touchdown PCR with designed primers. Then its unknown flanking sequences of the 5'- and 3'-ends were finally characterized by inverse PCR and nested PCR. Sequence analysis showed that the tdh gene contain 570 bp ORF which encoded 189 amino acids. The deduced amino acid sequence of the ORF was in significant homology with several Vibrio TDH. The product that the tdh gene expressed in E. coli was purified by Ni(2+)-IDA Sepharose affinity column. The activity of purified TDH was 4651 U mg(-1) protein by hide powder azure digestion. The lethal toxicity test showed that LD(50) values of the purified TDH were 5.68 and 8.34 microg TDH g(-1) body weight for mouse and crimson snapper, respectively. The complete ORF of tdh gene was obtained by touchdown PCR, inverse PCR and nested PCR. The ORF was perfectly expressed in E. coli. The activity and toxicity assays showed that the N-terminal signal peptide was essential to autocatalyse and fold correctly to obtain the activity and toxicity in the purified TDH. The Native-PAGE analysis showed that the activated tdh gene expressed in E. coli was a dimer with two identical subunits. This study demonstrates that the expressed activated TDH can produce the toxicity protein determined on mouse and fish, which will lead to better understandings of the identifying virulence factor that could be considered as a candidate antigen for vaccine and a diagnostic tool for vibriosis. Its use as an immunizing antigen might prevent the ability of V. alginolyticus to infect the marine aquatic animals, as a complementary measure to tick control and appropriate management in countries affected by vibriosis.

Expression of 25 hydroxyvitamin D3-1α-hydroxylase in human endometrial tissue

1,25(OH)2D3 (calcitriol) has been shown to play an important role in cell proliferation, differentiation and immune responsiveness. The enzyme responsible for calcitriol synthesis 25 hydroxyvitamin D3-1α-hydroxylase (1α-OHase) has been reported in many human tissues. The aim of this study was to investigate the expression of 1α-OHase in gynaecological tissues. Using a highly specific nested touchdown PCR we examined the expression of 1α-OHase in normal and malignant endometrial tissue and in human endometrial Ishikawa cells. In addition, we analyzed the protein expression of 1α-OHase by Western blot. The expression of 1α-OHase in normal and malignant endometrial tissue and Ishikawa cells was detected and splice variants of the enzyme in Ishikawa cells were identified. These data suggest an alternative splicing of 1α-OHase in malignant endometrial tissue and cells. We postulate that the expression of 1α-OHase gene variants may contribute to the antiproliferative effects of calcitriol. In conclusion, the modulation of the 1α-OHase opens up a new target for vitamin D3 related therapies in endometrial cancer.

Comparison of Single and Touchdown PCR Protocols for Detecting Pneumocystis jirovecii DNA in Paraffin‐Embedded Lung Tissue Samples

Comparison of Single and Touchdown PCR Protocols for Detecting <i>Pneumocystis jirovecii</i> DNA in Paraffin‐Embedded Lung Tissue Samples

Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax

BackgroundPlasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites.ResultsWe describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs) sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot.The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins.ConclusionWe identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains.

Optimized Adaptor Polymerase Chain Reaction Method for Efficient Genomic Walking

Genomic walking is one of the most useful approaches in genome-related research. Three kinds of PCR-based methods are available for this purpose. However, none of them has been generally applied because they are either insensitive or inefficient. Here we present an efficient PCR protocol, an optimized adaptor PCR method for genomic walking. Using a combination of a touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol achieves high sensitivity with low background noise. By applying this protocol, the insertion sites of a gene trap mouse line and two gene promoters from the incompletely sequenced Xenopus laevis genome were successfully identified with high efficiency. The general application of this protocol in genomic walking was promising.

Early detection of Schistosoma mansoni infection by touchdown PCR in a mouse model

A detection assay for Schistosoma mansoni DNA in mouse serum samples based on touchdown PCR was developed and evaluated. The serum samples could be assayed directly without the need to extract DNA. No cross reactions between S. mansoni and related species inducing human schistosomiasis were observed. After the infection, mouse sera and feces were collected for 8 weeks. Anti-worm antigen IgG and anti-soluble egg antigen IgG were detected in the sera at 6 weeks post-infection by ELISA. The parasite's eggs were detected in the feces at 8 weeks. In contrast, S. mansoni DNA was detected in the sera at 2 weeks post-infection. These data suggest that touchdown PCR is a potential tool for the early diagnosis of S. mansoni infection.

Presentation of splice variants of 1-alpha-hydroxylase in MCF-7 breast cancer cells

20097 Background: Calcitriol (1,25-dihydroxyvitamin D3) is the most active metabolite of vitamin D. It is known for its antiproliferative and differentiating effects on many cell populations including MCF-7 and other breast cancer cells. 1α-hydroxylase (CYP27B1) one of the key enzymes has been found in breast cancer cells suggesting an autocrine regulation of producing calcitriol. An alternative splicing of the encoding genes for the1α-hydroxylase might play a role in regulating enzyme levels and can explain tissue specific variations of the enzyme activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. Methods: In order to obtain more information on the abundance of 1α-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The protein products of the full length enzyme and its slice variants were confirmed by Western Blot. Results: The full length sequence of 1α-hydroxylase and two different splice variants including intron 1 were isolated. Conclusion: We hypothesize that the expression of the splice variants can lead to a quantitatively lower expression the mRNA of the full length enzyme. The abundance of less active 1α-hydroxylase protein products can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues. No significant financial relationships to disclose.

Relationship of trichloroethylene-induced medicamentosa like dermatitis to HLA-DMA and HLA-DMB

To investigate the susceptibility of trichloroethylene-induced medicamentosa like dermatitis by comparing the frequency of HLA-DMA and HLA-DMB in patients with trichloroethylene-induced medicamentosa like dermatitis and in normal controls. The DNA of lymphocytes in 61 patients with trichloroethylene-induced medicamentosa like dermatitis and in 60 people as the normal control were abstracted by using touchdown PCR amplification of HLA-DMA and HLA-DMB. Then through restriction fragment length polymorphism (RFLP) and sequence base typing, the alleles and genotypes were confirmed. The frequency of HLA-DMA and HLA-DMB in the two groups was compared. The HLA-DMA*0101 allele frequency in patients with trichloroethylene-induced medicamentosa like dermatitis was significantly higher than in the control group (71.3% vs 55.0%, P < 0.05). The allele frequency of HLA-DMA*0103 was significantly higher in the patient group than in the control group (11.5% vs 3.3%, P < 0.05). The ratio of *0102 homozygotes of HLA-DMA*0102 in the patient group was significantly higher than in the control group (25.0% vs 8.2%, P < 0.05). The ratio of *0102 heterozygotes of HLA-DMB*0101 in the patient group was lower than in the control group (P < 0.05). The polymorphisms of DMA may be related to the susceptibility of the patients with trichloroethylene-induced medicamentosa like dermatitis.

Chlamydia Pneumoniae in Asymptomatic Carotid Atherosclerosis

We evaluated, in 415 patients with asymptomatic carotid atherosclerosis: (i) the prevalence of C. pneumoniae DNA in atherosclerotic carotid plaques and peripheral blood mononuclear cells (PBMC); (ii) the distribution of C. pneumoniae in atherosclerotic carotid plaques and PBMC from the same patients; (iii) the correlation between circulating anti-chlamydial antibodies and the presence of C. pneumoniae DNA. Overall, 160 atherosclerotic carotid plaques and 174 PBMC specimens from patients with asymptomatic carotid atherosclerosis were examined by ompA nested touchdown PCR for presence of C. pneumoniae. In addition, C. pneumoniae DNA was detected in 81 specimens of atherosclerotic carotid plaque and PBMC obtained from the same patients. C. pneumoniae DNA was found in 36.9% of atherosclerotic carotid plaques and in 40.2% of PBMC specimens examined (P=NS). With regard to 81 patients, C. pneumoniae DNA was detected in 27.2% of atherosclerotic carotid plaques and in 44.4% of PBMC specimens(P=0.05). In 18 patients, the presence of C. pneumoniae DNA in PBMC specimens and atherosclerotic carotid plaques coincided (P=0.005). No statistically significant association was found between anti-C. pneumoniae antibodies (IgG and IgA) and positive PCR results. In conclusion, our results suggest that the detection of C. pneumoniae DNA in PBMC specimens seems to be a first-choice method to identify the patients at risk for endovascular chlamydial infection.

Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein

Chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. We used touchdown PCR to clone the chitinase (Schi) gene from Bacillus thuringiensis serovar sotto (Bt sotto) chromosomal DNA. Our DNA sequencing analysis revealed that the Bt sotto Schi gene consists of an open reading frame (ORF) of 2067 nucleotides with codes for the chitinase precursor. We also found that the putative promoter consensus sequences (the -35 and -10 regions) of the Bt soto Schi gene are identical to those of the chiA71 gene from Bt Pakistani, the chiA74 gene from Bt kenyae and the ichi gene from Bt israelensis. The Schi chitinase precursor is 688 amino acids long with an estimated molecular mass of 75.75 kDa and a theoretical isoelectric point of 5.74, and contains four domains, which are, in sequence, a signal peptide, an N-terminal catalytic domain, a fibronectin type III like domain and a C-terminal chitin-binding domain. Sequence comparison and the evolutionary relationship of the Bt sotto Schi chitinase to other chitinase and chitinase-like proteins are also discussed.

Combining Restriction Digestion and Touchdown PCR Permits Detection of Trace Isoforms of Histamine H 3 Receptor

The conserved sequences of the mouse histamine H3 receptor at the potential alternative splice junctions suggest that the splice isoforms found in guinea pig, rat, human, and hamster may also be present in the mouse. However, the trace amount isoforms are hard to be detected by the regular PCR approach. In this paper, we report a method in which the unspliced long isoform is cut by restriction endonuclease so that the short isoforms can be amplified to detectable levels to confirm the existence of the splice isoforms of H3 receptor mRNA in the mouse. This method is applicable to the detection of trace amounts of splice isoforms that coexist with the long, more abundant isoforms.

Assessment of Minimal Residual Disease (MRD) by Qualitative RT-PCR (Q-PCR) vs Real-Time Quantitative RT-PCR (RQ-PCR) in a Phase II Study of Acute Promyelocytic Leukemia (APL).

Assessment of MRD in APL by Q-PCR analysis for the presence or absence of PML-RARα mRNA after the completion of consolidation therapy in treatment regimens employing all-trans retinoic acid (ATRA) and chemotherapy (CT) has provided a reasonably accurate but not infallible prognostic test for disease recurrence (DR). This assessment has been made at detection sensitivity levels up to 1 in 104, which is considered to have optimal predictive value, since non-quantified lower levels of PML-RARα mRNA may not be relevant to clinical outcome. In the current study, we compared the results of Q-PCR (touchdown PCR for 40 cycles followed by post-PCR blot hybridization with a RARα chemiluminescent probe) to results from RQ-PCR in 15 patients on a Phase II clinical trial designed to eliminate CT (low-risk/LR patients, WBC <10K) or minimize CT (high-risk/HR patients, WBC >10K), using combined ATRA and arsenic trioxide therapy. All patients achieved complete remission (CR) and have been followed for a median of 1.2 years. DR occurred in 0/8 LR, 0/1 unclassified and 2/6 HR patients. At CR, all Q-PCR and RQ-PCR assays were positive. For RQ-PCR, the normalized quotient (NQ) value of PML-RARα, i.e., the PML-RARα copy number divided by the housekeeping gene glyceraldehyde-3′-phosphate dehydrogenase (GAPDH) copy number, ranged widely from 1.5 x 10−6 to 7.0 x 10−3. Post-CR follow-up data were:Patient GroupNumberQ-PCR+RQ-PCR+NQ RangeLR/Unclassified90/2416/249 x 10−9 – 1 x 10−7HR/no DR40/238/232 x 10−9 – 3 x 10−7HR/DR21/75/71 x 10−8 – 5 x 10−5For the 2 HR/DR patients, the last pre-DR results were, respectively: Q-PCR, positive and negative; RQ-PCR, NQ = 5 x 10−5 and NQ = 2.3 x 10−7. In overall comparisons to RQ-PCR results, all Q-PCR assays were positive for NQ >10−5, 3/4 were positive in the NQ 10−6 to 10−5 range, and all were negative for NQ <10−6. Additionally, dilution-reconstruction experiments confirmed the stochastic nature of PCR assays near the detection limit and indicated that RQ-PCR was about 10-fold more sensitive than Q-PCR. Although larger studies are needed, our results suggest that HR patients who are Q-PCR-negative but RQ-PCR-positive in the >10−7 – <10−5 range at a critical post-CR checkpoint may be at increased risk of DR and, more generally, they suggest that the results of RQ-PCR testing at a critical checkpoint can be used in combination with clinical risk assessment to stratify the intensity of subsequent MRD monitoring in individual patients.

The human ABCG1 gene: identification of LXR response elements that modulate expression in macrophages and liver

The ABC transporter ABCG1 (ATP binding cassette transporter G1), expressed in macrophages, liver, and other tissues, has been implicated in the efflux of cholesterol to high density lipoprotein. The ABCG1 gene is transcriptionally activated by cholesterol loading and activators of liver X receptors (LXRs) and retinoid X receptors (RXRs) through genomic sequences that have not been fully characterized. Here we show that ABCG1 mRNA is induced by LXR agonists in RAW264.7 macrophage cells, HepG2 hepatoma cells, and primary mouse hepatocytes. We identify two evolutionarily highly conserved LXR response elements (LXREs), LXRE-A and LXRE-B, located in the first and second introns of the human ABCG1 gene. Each element conferred robust LXR-agonist responsiveness to ABCG1 promoter-directed luciferase gene constructs in RAW264.7 and HepG2 cells. Overexpression of LXR/RXR activated the ABCG1 promoter in the presence of LXRE-A or LXRE-B sequences. In gel-shift assays, LXR/RXR heterodimers bound to wild-type but not to mutated LXRE-A and LXRE-B sequences. In chromatin immunoprecipitation assays, LXR and RXR were detected at LXRE-A and -B regions of DNA of human THP-1 macrophages. These studies clarify the mechanism of transcriptional upregulation of the ABCG1 gene by oxysterols in macrophages and liver, two key tissues where ABCG1 expression may affect cholesterol balance and atherogenesis.

Complete deficiencies of complement C4A and C4B including 2-bp insertion in codon 1213 are genetic risk factors of systemic lupus erythematosus in Thai populations

The complement component C4 is encoded by two genes: C4A and C4B on human chromosome 6p in the major histocompatibility complex (MHC). Most studies have linked the deficiencies in C4 with systemic lupus erythematosus (SLE) in Angio-Irish, North American, Black American, Mexican American, Australian and Japanese populations. Null alleles at either locus (C4AQ0 or C4BQ0) are relatively common in Americans occurring at the C4A and C4B loci in approximately 10% and 16% of normal individuals, respectively. In the present study, we extensively examined the possible association between homozygous C4Q0 and SLE in a large cohort of Thai populations diagnosed as SLE and further attempted to identify the genetic basis of C4Q0. One hundred and eighteen cases of SLE patients and 145 matched controls were genotyped by touchdown PCR. The results confirmed the previous studies that 5.93% (7/118) of C4 null genes: 2.54% (3/118) of C4AQ0 and 3.39% (4/118) of C4BQ0 were found in SLE patients. In contrast to other studies, we found no cases of C4 null genes in normal control (0 from 145 samples). To further investigate the genetic basis of C4 deficiency, all genomic DNAs were also analyzed for 2-bp (TC) insertion at codon 1213 in exon 29 which is a common mutation in many C4A null genes and a novel 1-bp deletion (C) at codon 522 in exon 13 that is common in most C4B null genes. Both mutation results in a flame-shift mutation and premature stop codon using sequence specific primers PCR (SSP-PCR) and direct sequencing. The results showed that there was 2-bp insertion in exon 29 of mutant C4B gene in one SLE patient carrying C4AQ0. There was no 2-bp insertion in exon 29 of both C4A and C4B genes in normal individual and the rest of SLE patients. All patients with C4AQ0 exhibited more than 5 ACR criteria including malar rash, oral ulcers, renal disorder, immunological disorder, anti-nuclear antibody, without hematological disorder. In contrast, all of C4BQ0 SLE patients showed 5 or 6 ACR criteria including hematological disorder, malar rash, oral ulcers, renal disorder, immunological disorder and anti-nuclear antibody. A patient who possesses C4AQ0 and 2-bp insertion in exon 29 of mutant C4B showed 9 ACR criteria but no discoid rash and hematological disorder. In conclusion, both C4AQ0 and C4BQ0 are the strong predisposing factors for SLE in Thais. It was supported by the absence of either C4A or C4B deletion in healthy control. We suggested that the different racial and genetic backgrounds could alter the thresholds for requirement of C4A or C4B protein levels in immune tolerance and regulation.

Use of Touch-Down Polymerase Chain Reaction to Enhance the Sensitivity of Mycobacterium Bovis Detection

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis-positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.

Close genetic relationship of early Neolithic cattle from Ziegelberg (Freising, Germany) with modern breeds

In 2003 a variety of crafts and bone specimens were found during excavations of a Neolithic settlement near Freising, the southernmost site of the Linear Pottery Culture in Bavaria. Six cattle bones were used to extract ancient DNA (aDNA). Applying nested and touchdown PCR, two fragments of the mitochondrial (mt) DNA control region could be amplified from specimen 533/III which yielded a total of 230 base pairs (bp). The sequence was compared with the homologous part of 40 extant breeds of Bos taurus and B. indicus and related species, such as Banteng (B. javanicus), Gaur (B. gaurus), the European bison (Bison bonasus) and the aurochs (B. primigenius). A neighbour joining tree was constructed based on the appropriate model of sequence evolution. The control region sequence of the 533/III cattle bone, whose age was determined by radiocarbon dating, clusters close to the extant European breeds, but distinctly apart from the basal aurochs and far distant from the B. indicus group. The archaeological and genetic analyses of Bos Ziegelberg demonstrate that domesticated cattle reached southern Bavaria at least 7000 years ago.