Mosaic and polymorphic imprinting of the WT1 gene in humans.

We have examined the imprinting of the Wilms' tumour suppressor gene (WT1) in human tissues. We confirm that WT1 is biallelically expressed in the kidney, however, in five of nine preterm placentae WT1 was expressed largely or exclusively from the maternal allele. Monoallelic expression of WT1 was also found in two fetal brains. These data demonstrate that WT1 can undergo tissue specific imprinting. Furthermore, because monoallelic expression of WT1 was not found in all placentae examined, WT1 imprinting may be genetically polymorphic within the human population.

Establishment of functional imprinting of the H19 gene in human developing placentae.

We have found that the imprinted H19 gene can be expressed either biallelically or monoallelically in the developing human placentae. H19 biallelic expression is confined to the placenta until 10 weeks of gestation, after which it becomes exclusively maternal, and does not affect allele-specificity or levels of IGF2 expression. The promoter region of H19 is hypomethylated at all stages of placental development, while the 3' portion shows progressive methylation of the paternal allele with gestation. Our observations demonstrate that the establishment of functional H19 imprinting occurs during the early development of the placenta and provide an opportunity to understand the mechanism by which the H19 primary imprint is manifested in somatic cells.

Simvastatin enhances bone formation around titanium implants in rat tibiae.

Statins are cholesterol-lowering drugs that have been reported to promote bone formation. The purpose of this study was to investigate the effect of simvastatin on the enhancement of bone formation around titanium implants. Thirty-week-old female rats received pure titanium implants in both tibiae. The animals were intra-peritoneally administered 0, 0.125, 1, 5 or 10 mg kg(-1) of simvastatin daily. After 30 days, the animals were sacrificed, and specimens were prepared. The bone contact ratio of the implant, bone density in the medullary canal and percentage of cortical bone were obtained. Markers for bone turnover were also measured using sera collected at the time of euthanasia. In the medullary canal, a scanty amount of bone was observed in the 0, 0.125 and 1 mg kg(-1) groups. In contrast, in both the 5 and 10 mg kg(-1) groups, thicker bone trabeculae were abundant. Histometric observations showed that the bone contact ratio and the bone density of both groups were significantly greater than those of the other groups (anova, P < 0.01). However, no significant difference in the percentage of cortical bone was found between groups. Serum chemistry showed that statin increased bone formation markers and decreased bone resorption markers. In conclusion, although the dose equivalent to that used in human patients with hypercholesterolemia was not effective, a simvastatin dose of 5 mg kg(-1) or higher increased medullary bone formation around the titanium. In contrast, no effect of simvastatin on pre-existing cortical bone was indicated.

Impact of surface contamination of implants with saliva during placement in augmented bone defects in sheep calvaria.

Our aim was to try and find out whether contamination with saliva during insertion of dental implants affects osseointegration in bone that has been augmented with different grafts. Six bony defects were created in each of the calvaria of six sheep, and then augmented with three different materials (autogenous bone, bovine bone, and resorbable biphasic ceramic bone substitute) After five weeks of healing, three implants contaminated with saliva (contaminated group) and three not contaminated (uncontaminated group) were placed in the centre of the augmented areas. For histomorphometric analysis, bone implant contact, bone area fraction occupancy, bone and material area, and bony area were measured after a healing period of five weeks. There was a significant difference between the contaminated and uncontaminated groups (p=0.036) for bone implant contact only in the augmented areas, but there were no significant differences in bone area fraction occupancy, bone and material area, and bony area. We conclude that contamination with saliva during placement of dental implants can significantly compromise bone implant contact in augmented areas, but had no significant effect on the formation of bone in areas more distant from the surface of the implant. We suggest that salivary contamination should be avoided during placement of dental implants in augmented areas.

Impact of salivary contamination during placement of implants with simultaneous bony augmentation in iliac bone in sheep.

Our aim was to investigate the possible impact of contamination with saliva on osseointegration during placement of implants with simultaneous bone augmentation. Six hemispheric shape bone defects (8mm in diameter×4mm deep) were prepared in each iliac bone of six sheep. A dental implant (2.9mm in diameter×10mm long) was placed in the centre of each defect, and then pairs of defects were filled with one of the following bone augmentation materials: autogenous bone, autogenous bone plus bovine bone, or resorbable biphasic ceramic bone substitute. One site in each augmentation group was impregnated with saliva (contaminated group), while the other was not (non-contaminated group). Bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO) within implant threads were measured after a healing period of five weeks, both in respect of the implant inserted in the augmented bone and in that inserted in the residual bone. Overall results showed that there was a significant difference between the contaminated and non-contaminated group for BIC in the augmented implant (p=0.028), while there were no significant differences in the implant in residual bone (p=0.722). For BAFO, there were no significant differences between the contaminated and non-contaminated groups among the different augmentation materials. The results showed that contamination with saliva during placement of an implant with simultaneous bone augmentation had a serious deleterious effect on osseointegration of the aspect of the implant within the augmented defect. Contamination with saliva during placement of an implant with simultaneous bone augmentation should therefore be avoided.

Cellular localization of placenta-specific human endogenous retrovirus (HERV) transcripts and their possible implication in pregnancy-induced hypertension.

We previously investigated tissue specificity and temporal changes in expression of five human endogenous retroviruses (HERVs), including syncytin/ERVWE1 and syncytin 2. In the current study, we examined the cellular localization and quantified the transcripts of five HERVs, syncytin, syncytin 2, HERV-H7/F(XA34), HERV-Fb1, and HERV-HML6-c14, in order to elucidate their physiological and etiological roles in the placenta and in pregnancy-induced hypertension (PIH), respectively. In situ hybridization revealed trophoblast-specific transcription of all five HERVs. Consistent with a previous immunohistochemical analysis, syncytin 2 transcripts were detected only in cytotrophoblasts. All the HERVs except HERV-HML6-c14 (HML6-c14) were predominantly localized in the cytoplasm of syncytiotrophoblasts and/or cytotrophoblasts. Quantitative analysis showed that transcriptional levels of these four HERVs were lower in placentas obtained from pregnant women with PIH (n=22) than in those from normotensive pregnant women (n=87) and that the differences were statistically significant (p=0.001, 0.01, <0.001, and 0.04 for syncytin, syncytin 2, HERV-H7/F(XA34), and HERV-Fb1, respectively). In contrast to the other HERVs, HML6-c14 transcripts localized to the nucleus and the average transcriptional level of HML6-c14 was higher in PIH placentas than in control placentas from normotensive pregnant women, although the differences did not reach significance (p=0.19). These results suggest that placenta-specific HERVs may have some function in the human placenta and that their reduced expression in PIH placentas is not merely a secondary effect derived from the pathology of PIH placentas.

MEFV mutation analysis of familial Mediterranean fever in Japan.

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of fever with serosal inflammation. FMF gene (MEFV) mutations have been identified primarily in patients from Mediterranean populations. Although several clinical cases have been reported in Japan, there have been few reports to date on mutation analysis. We studied FMF patients and their relatives to examine the clinical and genetic features of this disease in the Japanese population. METHODS: Twelve Japanese FMF patients who met the Tel Hashomer criteria and a total of 17 relatives from 5 of 10 families underwent molecular genetic studies to detect MEFV mutations. The characteristics of these Japanese FMF patients and geno-phenotypical correlations were examined. RESULTS: Almost all of our patients had been suffering for a long time from fever of unknown origin and one patient also had systemic amyloidosis. In our 12 FMF patients, we detected the substitutions E84K, L110P, E148Q, R761H and M694I. We also newly diagnosed 2 relatives as having FMF based on clinical symptoms and the existence of FMF mutations. One patient was homozygous for E148Q, the patient with systemic amyloidosis was a homozygote for M694I and 4 patients from 3 families were compound heterozygotes for E148Q and M694I. Three patients in one family were compound heterozygotes for E148Q, L110P and M694I. There were 3 patients who were heterozygous for E84K, L110P-E148Q or M694I and had no other nucleotide changes in the exons of MEFV. On the other hand, 2 relatives who had never experienced symptoms of FMF were homozygous for L110P-E148Q as well as compound heterozygous for E148Q/E148Q-R761H. E148Q and M694I were the most frequently detected substitutions in our study. CONCLUSIONS: MEFV mutations occur in Japanese FMF patients though FMF is rare in Japan. The identification of MEFV mutations could be a reliable diagnostic test for FMF. The results of genetic analyses on 14 Japanese FMF patients in this study revealed that E148Q and M694I are frequent alleles.

Modulation of epidermal growth factor receptor proto-oncogene transcription by a promoter site sensitive to S1 nuclease.

The epidermal growth factor (EGF) receptor is the functional target of the mitogen EGF and the cellular homolog of the avian erythroblastosis virus erbB oncogene product. Regulation of expression of the proto-oncogene encoding the EGF receptor can be elucidated by studying the structure and function of the gene promoter outside the confines of the cell. Previously, we reported the isolation of the human EGF receptor gene promoter. The promoter is highly GC rich, contains no TATA or CAAT box, and has multiple transcription start sites. An S1 nuclease-sensitive site has now been found 80 to 110 base pairs (bp) upstream from the major in vivo transcription initiation site. Two sets of direct repeat sequences were found in this area; both conform to the motif TCCTCCTCC. When deletion mutations were made in this region of the promoter by using either Bal 31 exonuclease or S1 nuclease, we found that in vivo activity dropped three- to fivefold, on the basis of transient-transfection analysis. Examination of nuclear protein binding to normal and mutated promoter DNAs by gel retardation analysis and DNase I footprinting revealed that two specific factors bind to the direct repeat region but cannot bind to the S1 nuclease-mutated promoter. One of the specific factors is the transcription factor Sp1. The results suggest that these nuclear trans-acting factors interact with the S1 nuclease-sensitive region of the EGF receptor gene promoter and either directly or indirectly stimulate transcription.

Amplification of human argininosuccinate synthetase pseudogenes.

The human genome contains multiple pseudogenes for an argininosuccinate synthetase (AS) gene. To elucidate the molecular mechanisms of generation and dispersion, complete nucleotide sequences of four different AS pseudogenes, psi AS-Y, psi AS-A1, psi AS-A2 and psi AS-A3, have been determined. A comparison of these sequences with those of three reported AS pseudogenes, psi AS-1, psi AS-3 and psi AS-7 revealed that two pairs, psi AS-Y/psi AS-7 and psi AS-A3/psi AS-1, are highly homologous but not identical, thereby suggesting that one of the pairs is generated by a duplication of the other member of the pairs. The psi AS-Y, which is probably located on chromosome Y, and the partially sequenced psi AS-7 are both interrupted by an Alu element at exactly the same site in their 3'-end regions. These two Alu elements are located in an opposite orientation relative to the direction of transcription of the pseudogene, and their possible role on pseudogene dispersion was examined. The psi AS-A1 is also accompanied by an Alu element at its 3' end. In this case, the orientation of the Alu element is the same as that of the pseudogene. The psi AS-A1 and the Alu element are flanked with direct repeats, as if they had been inserted into a chromosomal site, as a single unit.

Augmented expression of secondary lymphoid tissue chemokine and EBI1 ligand chemokine in Crohn's disease.

BACKGROUND: A dominant T helper type 1 (Th1) immune response is thought to be involved in Crohn's disease (CD). SLC/CCL21 and ELC/CCL19, chemokines that regulate T cell homing and promote recirculating T and dendritic cell (DC) interactions, help control antigen specific T cell responses. AIMS: To investigate the Th1 response and SLC and ELC in CD pathogenesis. METHODS: Surgically resected intestine and mesenteric lymph nodes (MLNs) from controls and patients with CD and ulcerative colitis (UC) were investigated. CD3, CD83, HECA452, VEGFR3, SLC, ELC, and CCR7 expression was studied immunohistochemically. CCR7 mRNA was quantified using real time RT-PCR. RESULTS: ELC was almost undetectable in intestinal samples. SLC was found sporadically in lymphoid follicles, lymphoid aggregate venules, and lymphatic vessels. In MLNs, SLC was highly expressed in high endothelial venules (HEVs), lymphatic vessels, and stromal DCs, predominantly in T cell areas. ELC was highly expressed in mature DCs. There were significantly more SLC positive HEVs and ELC positive mature DCs, important components of T cell areas, in CD. SLC, ELC, and CCR7 mRNA was significantly higher in CD MLNs compared with UC. CD MLNs had increased expression of SLC and ELC, mainly in HEVs, mature DCs, and lymphatic vessels, inducing T cell hyperplasia. CCR7 mRNA was increased in T cell areas. CONCLUSION: The dominant Th1 immune response is facilitated by interaction of SLC positive HEVs/lymphatic vessels, ELC positive mature DCs, and CCR7 positive T cells in hyperplastic T cell areas. In CD, memory T cells and mature DCs may home to MLN.

A histologic study on the fate of intradermally implanted epidermal cells in guinea pigs: a new method for evaluation of skin allograft survival.

The fate of allogeneic (strains 13, 2, and JY-1) and autologous epidermal cell (EC) suspensions injected intradermally was investigated histologically in JY-1 strain guinea pigs. Epidermal cells were found to proliferate actively in the dermis and form EC nests with central keratinization. The significant reject reaction associated with necrosis of the epidermal structures was seen in due time in the animals implanted with allogeneic ECs. We attempted to assess the effect of cyclosporin A (CYA) on skin allograft survival by observing the fate of strain 13 ECs implanted intradermally into the CYA-treated JY-1. Successful prolongation of allograft survival with CYA was clearly demonstrated by this method. This is considered to be a useful experimental way for evaluation of skin allograft survival and to be suitable for routine use.

Are cerebrovascular factors involved in Alzheimer's disease?

Recent epidemiological studies have shown that vascular risk factors may be involved in Alzheimer's disease (AD) as well as dementia in general. To investigate the relation between a vascular disorder and AD pathology, current criteria are defective because most depend on exclusion of a cerebrovascular disorder. Epidemiological studies have indicated the possibilities that arteriosclerosis, abnormal blood pressure, diabetes mellitus and smoking may be related to the pathogenesis of AD. As for the mechanism that vascular disorders influence AD, it is presumed that amyloid deposition may be caused by a vascular disorder. Alternatively, a vascular event may cause progression of subclinical AD to a clinical stage. Insulin resistance and apolipoprotein E may also be involved in these mechanisms. Our studies show that ischemia-induced the Alzheimer-associated gene presenilin 1 (PS1) and endoplasmic reticulum-stress, generated from a vascular disorder, may unmask clinical AD symptoms caused by presenilin mutation, suggesting that a vascular factor might be involved in the onset of familial AD.

Characterization of the gene encoding human pituitary-specific transcription factor, Pit-1.

Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone- and prolactin-encoding genes. A chromosomal gene related to human Pit-1 isolated from human gene libraries was over 14 kb long and split into six exons. All of the splice donor and acceptor sites conformed to the GT/AG rule. The gene was mapped to human chromosome region 3p11.

Identification, characterization and mapping of the human ZIS (zinc-finger, splicing) gene.

From a human fetal brain cDNA library, we isolated two transcripts (ZIS-1 and ZIS-2) corresponding to the human ZIS gene, an ortholog of the rat Zis (zinc finger, splicing). A comparison of base sequences of the cDNA and its corresponding genomic DNA (a P1-derived artificial chromosome clone) revealed that both transcripts have an ORF of 1011bp and encodes 337 amino acids, but ZIS-1 has 10 exons and ZIS-2 contains 11 exons. Although both transcripts share the first nine exons, exon 10 of ZIS-2 is lacking in ZIS-1, and instead, exon 11 (10th exon) of ZIS-1 is larger in size, leading to the longer 3'-UTR. Thus, the two transcripts result from differential splicing. A Northern blot analysis on various adult and fetal tissues revealed that 5.2- and 3.2-kb transcripts were ubiquitously expressed, and 3.9- and 1.9-kb transcripts were highly expressed in the fetal brain and kidney, respectively. There were several other transcripts that may be alternatively processed forms of the human ZIS. Considering the ZIS gene size, the 3.2-kb transcripts most likely corresponds to ZIS-1 and may act as a major transcript of ZIS. The human ZIS has a high homology to the rat Zis for the coding DNA sequence with 91% identity and for the amino acid sequence with 87% identity. ZIS and Zis contain the same numbers of exons and introns. Both genes have unusually long 3'-UTR, and their encoding proteins contain similar components, i.e. a zinc finger domain, a nuclear localization signal, an Asp-Glu region, and a Ser-Arg-rich region. Furthermore, the expression patterns of the two genes in tissues are similar each other. Thus, the human ZIS may act as a transcriptional factor to regulate transcription and/or splicing, as does the rat Zis.

Vasorelaxant mechanism of KRN2391 and nicorandil in porcine coronary arteries of different sizes.

1. The relaxant mechanisms of action of KRN2391, a novel vasodilator, and nicorandil on epimyocardial coronary artery (2.5- 3.0 mm outer diameter) and mid-myocardial coronary artery (0.8-1.0 mm outer diameter) were investigated in porcine isolated coronary arteries. In addition, the vasorelaxant responses of KRN2391 and nicorandil were compared with those of nitroglycerin and cromakalim, a K+ channel opener, in epi- and mid-myocardial coronary arteries. 2. Nitroglycerin showed a more potent relaxant effect on epi-myocardial coronary arteries than on mid-myocardial coronary arteries, whereas cromakalim produced greater relaxation responses in mid-myocardial coronary arteries. There was no difference between epi- and mid-myocardial coronary arteries in terms of the relaxant effect of KRN2391 and nicorandil. 3. Relaxation induced by KRN2391 in epi- and mid-myocardial coronary arteries was inhibited by oxyhaemoglobin, a pharmacological antagonist of nitrovasodilators, and glibenclamide, a pharmacological antagonist of K+ channel opening drugs. However, the inhibitory effect of glibenclamide on KRN2391-induced relaxation was greater in mid-myocardial coronary artery than in epi-myocardial coronary artery. 4. Relaxation induced by nicorandil was inhibited by oxyhaemoglobin alone in epi-myocardial coronary arteries and by both oxyhaemoglobin and glibenclamide in mid-myocardial coronary arteries. 5. In epi- and mid-myocardial coronary arteries, relaxation induced by cromakalim was inhibited by glibenclamide but not by oxyhaemoglobin, whereas relaxation induced by nitroglycerin was inhibited by oxyhaemoglobin but not by glibenclamide. 6. These results suggest that KRN2391 and nicorandil exhibit a dual mechanism of action acting partly as a nitrate and partly as a K+ channel opener. The mechanism of action of these drugs depend on the segment of coronary artery studied. Furthermore, the dual mechanism of action of KRN2391 and nicorandil seems to contribute to the equipotent relaxant effect between epi- and mid-myocardial coronary arteries.

Contribution of cyclic GMP formation to KRN2391-induced relaxation in coronary artery of the pig.

1. In the present study, we investigated the relationship between relaxation and guanosine 3':5'-cyclic monophosphate (cyclic GMP) formation induced by KRN2391, compared with those induced by nicorandil and nitroglycerin, in the coronary artery of the pig. 2. KRN2391 (10(-8)-3 X 10(-5) M), nicorandil (10(-8)-3 X 10(-4) M) and nitroglycerin (10(-9)-10(-5) M) antagonized the contraction caused by 25 mM KCl in a concentration-dependent manner. 3. The concentration-relaxation curves for KRN2391, nicorandil and nitroglycerin shifted rightward in the presence of methylene blue (10(-5) M). 4. KRN2391 (10(-6) M), nicorandil (10(-4) M) and nitroglycerin (10(-6) M) induced an increased in cyclic GMP. 5. The magnitude of the shift of the concentration-relaxation curve caused by methylene blue and the increase in cyclic GMP with KRN2391 were lower than those with nicorandil and nitroglycerin. 6. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) level was not increased by KRN2391 even at a concentration that produced full relaxation. 7. The present results suggest that KRN2391-induced relaxation in the coronary artery of the pig is partly due to the increase in cyclic GMP formation through the stimulation of guanylate cyclase.

Excess functional copy of allele at chromosomal region 11p15 may cause Wiedemann-Beckwith (EMG) syndrome.

Wiedemann-Beckwith syndrome (WBS) is a genetic disorder with overgrowth and predisposition to Wilms' tumor. The putative locus of the gene responsible for this syndrome is assigned to chromosome region 11p15.5, and genomic imprinting in this region has been proposed: the paternally derived gene(s) at 11p15.5 is selectively expressed, while the maternally transmitted gene(s) is inactive. We examined 18 patients for the parental origin of their 11p15 regions. DNA polymorphism analyses using 6 loci on chromosome 11 showed that 2 patients with duplications of 11p15 regions from their respective fathers and one from the mother, indicating the transmission of an excessive paternal gene at 11p15 to each patient. Our results, together with the previous findings in karyotypically normal or abnormal patients and in overgrowth mouse experiments, are consistent with imprinting hypothesis that overexpression of paternally derived gene(s) at 11p15.5, probably the human insulin-like growth factor II (IGF-II) gene, may cause the phenotype. Total constitutional uniparental paternal disomy (UPD) or segmental UPD for the 6 loci examined of chromosome 11 was not observed in our 12 sporadic patients. In order to explain completely the inheritance of this syndrome in patients with various chromosomal constitutions, we propose an alternative imprinting mechanism involving the other locus that may be paternally imprinted and may suppress the expression of this gene.

Molecular and clinical study of 61 Angelman syndrome patients.

We analyzed 61 Angelman syndrome (AS) patients by cytogenetic and molecular techniques. On the basis of molecular findings, the patients were classified into the following 4 groups: familial cases without deletion, familial cases with submicroscopic deletion, sporadic cases with deletion, and sporadic cases without deletion. Among 53 sporadic cases, 37 (70%) had molecular deletion, which commonly extended from D15S9 to D15S12, although not all deletions were identical. Of 8 familial cases, 3 sibs from one family had a molecular deletion involving only 2 loci, D15S10 and GABRB3, which define the critical region for AS phenotypes. The parental origin of deletion, both in sporadic and familial cases, was exclusively maternal and consistent with a genomic imprinting hypothesis. Among sporadic and familial cases without deletion, no uniparental disomy was found and most of them were shown to inherit chromosomes 15 from both parents (biparental inheritance). A discrepancy between cytogenetic and molecular deletion was observed in 14 (26%) of 53 patients in whom cytogenetic analysis could be performed. Ten (43%) of 23 patients with a normal karyotype showed a molecular deletion, and 4 (13%) of 30 patients with cytogenetic deletion, del(15) (q11q13), showed no molecular deletion. Most clinical manifestations, including neurological signs and facial characteristics, were not distinct in each group except for hypopigmentation of skin or hair. Familial cases with submicroscopic deletion were not associated with hypopigmentation. These findings suggested that a gene for hypopigmentation is located outside the critical region of AS and is not imprinted.

Novel structure of the 5' end region of the human argininosuccinate synthetase gene.

The structure of the 5' end region of the human argininosuccinate synthetase (AS) gene was analyzed in detail. The 5' flanking region up to 350 nucleotides from the putative transcription initiation site is unusually G+C rich (80%). Three repeats of a CCGCCC sequence and one complementary GGGCGG sequence are present in this region. It contains no CCAAT box-like sequence, but does contain one typical TATA box and a 20 bases-long inverted repeat sequence that could form a stem and loop structure just upstream from the TATA box. An octanucleotide sequence, AGAAGTGA, found in the 5' flanking region of the AS gene is also commonly present in those regions of the two other human genes expressed mainly in the liver. These structural features of the AS gene indicate that it shares common structures with two completely different groups of genes, i.e. tissue-specific genes and housekeeping ones. Moreover, two enhancer core-like sequences, alternating purine-pyrimidines tracts and two independent Alu type highly repetitive sequences are apparent in the first intron of the AS gene.

A simple and efficient amplification method of DNA with unknown sequences and its application to microdissection/microcloning.

An alternative method for amplification of DNA with unknown sequences was developed. This involves the direct ligation of a primer oligodeoxyribonucleotide itself to restricted DNA fragments with unknown sequences to be amplified by PCR. The oligonucleotide need not be phosphorylated and need not be annealed with its complementary oligonucleotide in advance for ligation. The ligation reaction seems to be independent of the concentration of unknown DNA, proceeds in short time, and is efficient. The ligation efficiency was more than 30% at a low concentration, 10 fg/microliters, of DNA. This method was applied to a microdissection/microcloning of the short arm of human chromosome 2. Of 65 clones screened for the highly repetitive sequences with total human genomic DNA, eleven (17%) were positive. Their inserts ranged in size from 150 to 1,200 bp (average, 460 bp). In Southern blot analysis, thirty consecutive clones all detected signals common to both total human genomic DNA and mouse-human hybrid cell DNA containing only chromosome 2 of human origin. Among them, 24 (80%) were unique sequences, and 6 (20%) were multi-copy (or intermediate-repeat) sequences. Thus, this method is simple and efficient, and provides an alternative way to amplify unknown DNA.