Polytene chromosomes of Oxytricha: Biochemical and morphological changes during macronuclear development in a ciliated protozoan

After conjugation in the ciliated protozoan, Oxytricha, polytene chromosomes are formed during the development of a macronucleus from a micronucleus. Here we report a microscopic study of these chromosomes and an analysis of their DNA. The polytene chromosomes of Oxytricha bear a strong morphological resemblance to the polytene chromosomes of the Dipteran salivary gland. The nucleus of a developing macronuclear anlage contains 120±2 polytene chromosomes and each chromosome has an average of 81 bands; a total of about 10,000 bands per nucleus. At a later stage in development, the number of bands per chromosome is reduced by a factor of four, presumably due to fusion of adjacent bands. The polytene chromosomes then break up into their constituent bands, each of which is encased in a vesicle. There are about 2,700 vesicles per nucleus. — During the growth of polytene chromosomes, there is a change in the relative proportion of sequences in the DNA. The DNA from polytene nuclei has a buoyant density of 1.695 g/cc, significantly lighter than the density of the original micronuclear DNA (1.698 g/cc to 1.702 g/cc). We interpret this buoyant density change to be the result of differential replication of DNA sequences during polytene chromosome growth. A second change in DNA composition occurs after the polytene stage of development, shown by a shift in buoyant density to 1.701 g/cc in the DNA of the mature macronucleus. During this second process, the molecular weight of the DNA is reduced from greater than 50×10⁶ daltons to about 2×10⁶ daltons.This paper is No. VI in the series, DNA of Ciliated Protozoa.     

Evidence for dosage compensation in parthenogenetic Hymenoptera

Amount of DNA-Feulgen staining in individual somatic nuclei and mature sperm of the parthenogenetic wasps, Habrobracon juglandis, H. serinopae, and Mormoniella vitripennis, were determined with a scanning microdensitometer. The haploid genome for both species of Habrobracon was estimated to be 0.15–0.16×10^–12 g DNA, corresponding to a molecular weight of roughly 10×10¹⁰ daltons. The haploid genome of M. vitripennis is approximately twice this value, 0.33–0.34×10^–12 g, or about 20×10¹⁰ daltons. Measurements made on dividing nuclei from syncytial preblastoderm embryos of H. juglandis and M. vitripennis showed that the chromosomes of impaternate males were present in the haploid number and contained the C amount of DNA; whereas nuclei from female preblastoderm embryos contained the diploid number of chromosomes and the 2C amount of DNA. However, hemocyte and brain cell nuclei from either male or female adult wasps contained 2C and 4C amounts of DNA. Both sexes also showed equivalent levels of polyploidy (8C, 16C, or 32C) in Malpighian tubule nuclei. Therefore, in these parthenogenetic species, a mechanism must exist that compensates during later development for the initial two-fold difference in the chromatin content of somatic nuclei in haploid male and diploid female embryos. Hemocytes from impaternate Mormoniella diploid males and triploid females contain the 2C and 3C amounts of DNA, respectively. Therefore dosage compensation involves an additional cycle of DNA replication only in haploid cells, and it insures that a certain minimum quantity of DNA is received by each somatic cell.     

Organelle DNA accumulation in the recently evolved papaya sex chromosomes

Sex chromosomes are a pair of specialized chromosomes containing a sex determination region that is suppressed for recombination. Without recombination, Y chromosomes are thought to accumulate repetitive DNA sequences which contribute to their degeneration. A pair of primitive sex chromosomes controls sex type in papaya with male and hermaphrodite determined by the slightly different male-specific region of the Y (MSY) and hermaphrodite-specific region of Y^h (HSY) chromosomes, respectively. Here, we show that the papaya HSY and MSY in the absence of recombination have accumulated nearly 12 times the amount of chloroplast-derived DNA than the corresponding region of the X chromosome and 4 times the papaya genome-wide average. Furthermore, a chloroplast genome fragment containing the rsp15 gene has been amplified 23 times in the HSY, evidence of retrotransposon-mediated duplication. Surprisingly, mitochondria-derived sequences are less abundant in the X and HSY compared to the whole genome. Shared organelle integrations are sparse between X and HSY, with only 11 % of chloroplast and 12 % of mitochondria fragments conserved, respectively, suggesting that the accelerated accumulation of organelle DNA occurred after the HSY was suppressed for recombination. Most of the organelle-derived sequences have divergence times of <7 MYA, reinforcing this notion. The accumulated chloroplast DNA is evidence of the slow degeneration of the HSY.     

Mass characteristics of DNA obtained from chromosomes of a human carcinoma cell line

Sedimentation studies of DNA from chromosomes extracted from human mitotic cells showed that highmolecular-weight DNA can be obtained if cell hypotonic treatments and prolonged metaphase blocks are avoided. Two types of large double-stranded DNA were observed. One of these (M _r = 2.5×10⁸) appeared as a size class with characteristics reminiscent of the chromosomal DNA subunit hypothesis. However, this DNA is the decay product of larger molecules, whose minimum molecular weight is 6×10⁸.     

Chromosome-sized DNA molecules from Drosophila

Measurements of viscoelastic retardation times of detergent-Pronase lysates of Drosophila cells demonstrated the presence of large numbers of DNA molecules of a size commensurate with that of the chromosomes. The values estimated from the retardation times for the molecular weights of the largest molecules ranged from about 20×10⁹ to 80×10⁹ daltons depending on the species of Drosophila. The molecular weights of the DNA molecules were independent of the metaphase shapes (i.e., metacentric or submetacentric), but were proportional to the DNA contents of the chromosomes in the case of translocations or deletions. It was concluded, therefore, that the DNA molecules must run the length of the chromosome and cannot be discontinuous at the centromere. When compared with the values of the DNA contents of Drosophila chromosomes determined by other methods, the results were consistent with the model of one, or possibly two, DNA molecules per chromosome; the simplest conclusion, that there is only one DNA molecule per chromosome (for simple chromosomes), rests on a long extrapolation of an empirical relation between retardation time and molecular weight, but is also favored by indirect evidence. Further possibilities which could not be excluded were that the large DNA molecules contained Pronase-resistant, non-DNA links, or that a fraction of smaller DNA molecules might also have been present in the chromosomes. Chromosome-sized DNA molecules were obtained almost quantitatively from unsynchronized cultured cells, suggesting that the size of the chromosomal DNA is conserved throughout much of the cell cycle. The molecules were stable for periods of up to several days at 50° C in solutions containing detergent, Pronase, and EDTA.     

Strukturverändernde Wirkung von FUdR auf polytäne Chromosomen und Beziehungen zwischen Replikationsdauer und Bruchhäufigkeit von Querscheiben

Larvae of Chironomus th. thummi at the age of 10 hours after hatching were treated for 20 hours with 10^–4 M FUdR. The salivary gland chromosomes were studied at fourth instar. FUdR induces chromosomal constrictions and partial breakage of various diameters ranging from 1/2 to less than 1/16 of the total cross section of the polytene chromosomes. Breaks were predominantly found in chromosome regions containing bands of high DNA content. By H³-thymidine-autoradiography it is demonstrated that bands which are frequently broken are late replicating. This is shown by histograms correlating the distribution of breaks over the chromosomes with labeling patterns obtained in late S.—As bands with a great amount of DNA do not only replicate late but also spend the longest time in DNA synthesis, it is assumed that they also represent the largest replicons. It is discussed if this is the reason why FUdR induces breaks preferentialy in bands of high DNA content.     

Cryptic recombination in the ever-young sex chromosomes of Hylid frogs

Sex chromosomes are expected to evolve suppressed recombination, which leads to degeneration of the Y and heteromorphism between the X and Y. Some sex chromosomes remain homomorphic, however, and the factors that prevent degeneration of the Y in these cases are not well understood. The homomorphic sex chromosomes of the European tree frogs (Hyla spp.) present an interesting paradox. Recombination in males has never been observed in crossing experiments, but molecular data are suggestive of occasional recombination between the X and Y. The hypothesis that these sex chromosomes recombine has not been tested statistically, however, nor has the X‐Y recombination rate been estimated. Here, we use approximate Bayesian computation coupled with coalescent simulations of sex chromosomes to quantify X‐Y recombination rate from existent data. We find that microsatellite data from H. arborea, H. intermedia and H. molleri support a recombination rate between X and Y that is significantly different from zero. We estimate that rate to be approximately 10⁵ times smaller than that between X chromosomes. Our findings support the notion that very low recombination rate may be sufficient to maintain homomorphism in sex chromosomes.     

Location of messenger specifying sequences in mammalian chromosomes

In situ hybridization of complementary DNA (cDNA) synthesized from total cytoplasmic polyadenylated RNA isolated from Chinese hamster cells was employed to investigate the distribution of messenger specifying sequences on mammalian chromosomes. The kinetics of cDNA-nuclear DNA annealing indicate that about 85% of the cDNA represents sequences which are transcribed from non-repetitive DNA sequences. When cDNA is hybridized back to its template RNA, the reaction kinetics show that more than 60% of the poly(A) RNA is at least 10⁴ times more complex than rabbit globin mRNA. In situ hybridization of cDNA to Chinese hamster cells fixed on slides shows no significant clustering of silver grains on interphase nuclei. On metaphase chromosomes the majority of silver grains are localized in euchromatic areas. It appears that all euchromatic segments have similar grain densities. Chromosomes 1 and 2, which have relatively little heterochromatin, do not have a higher grain density than the other chromosomes. However, the Y chromosome, which is entirely heterochromatic, contains only about 1/3 the grain density of the chromosomes 1 or 2. — When the cDNA, which anneals only to the high abundancy class of poly(A) RNA was fractionated and hybridized in situ to Chinese hamster chromosomes, the distribution of silver grains is localized in the euchromatic areas. The Y chromosome and the heterochromatic arm of the X chromosome contain less grains; telomeres of some autosomes have higher grain densities. The oligo-(dT) primer in cDNA did not affect the results of this study since no grains are found when ₃H-poly(dT) was used as probe for in situ hybridization. The majority (>90%) of the grains could be blocked by competition with excess repetitive DNA in the hybridization reaction, indicating that the in situ hybridization involved predominantly repetitive sequences.     

Double-stranded DNA with the size expected of replicons can be released from Chironomus polytene chromosomes

The effect of the drug 5-fluorodeoxyuridine on DNA synthesis in Chironomus polytene chromosomes was investigated. The DNA was labelled by injection of radioactive precursor into living animals pre-treated with the drug, extracted with a neutral non-denaturing buffer at 25 ° C and then characterized by gel electrophoresis. After a short pulse a heterogeneous double-stranded DNA population is released from the polytene chromosome. These fragments are later joined together to produce a double-stranded DNA with a size ranging between 8–13 × 10⁶ D. The release of both types of fragments from the polytene chromosome is prevented by lysing the cells at 0 ° C instead of at 25 ° C. The larger double-stranded DNA has the size expected of replicons in Chironomus. The results are discussed in relation to the organization of replication in polytene chromosomes.     

Analysis of DNA of male and femaleSphaerocarpos donnellii Aust. (Liverwort)

Summary The karyotypes of females and males ofSphaerocarpos donnellii differ in that there is a large essentially heterochromatic X chromosome in the female (approx. 25 volume-% of the autosomes) and a small Y chromosome in the male (0.1–3 volume-% of the autosomes). DNA from females and males differ in buoyant densities in cesium chloride equilibrium gradients (1.702₅ and 1.703₅g cm^-3, respectively) and in melting points (87.5 and 88.5°C in SSC). The differences are statistically significant. Base analyses revealed 2.5 Mol-% of the rare base 5-methyl cytosine. Upon reassociationSphaerocarpos DNA behaves kinetically in a heterogeneous manner. About 22% of the DNA is repetitive with an average kinetic complexity of 1.6×10⁸ daltons. The kinetic complexity of the slow reassociating DNA fraction, considered to be of the single copy type, is 3.2×10¹⁰ daltons. No difference in the renaturation behavior between DNA of males and females could be detected with the techniques used. Our data thus indicate that X chromosomal DNA cannot contain large amounts of highly repeated nucleotide sequences and that it is slightly enriched in AT content compared to the autosomes.     

Somatic embryogenesis and plants from zygotic embryos of coconut (Cocos nucifera L.) in vitro

Complete plants were grown from zygotic embryos cultured on Y3 basal liquid medium supplemented with coconut milk, BA and NAA. Explants from stem, leaf and rachilla of mature coconut trees turned green and swelled on Y3 semi-solid basal media supplemented with 2,4-D, K, NAA, BA and activated charcoal. Callus was initiated in explants from the subapical regions of the stem on Y3 basal medium supplemented with 2,4-D (4.52×10²M). Globular embryo-like structures were obtained when this callus was subcultured to auxinless medium. Root formation was obtained from leaf explants on Y3 basal medium containing citric acid, ascorbic acid and 2,4-D (4.52×10² M). Globular embryo-like structures were also obtained directly from leaf explants on a Y3 basal medium supplemented with 2,4-D (2.26×10² M). Callus isolated from rachilla explants on Y3 basal medium containing 2,4-D(4.52×10² M), formed nodular structures when transferred to medium with 2,4-D (2.3×10¹ M). These nodules developed roots from the base of the nodular growth whereas from the upper portion shoots were observed on Y3 basal liquid medium.Abbreviations K kinetin - BA Benzyl adenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - CM Coconut milk - IAA Indole acetic acid - 2iP N⁶-r-r-dimethyl allyl amino purine NCL Communication No. 3471     

The mechanism of C-banding: depurination and β-elimination

C-banding of chromosomes involves the differential solubilization of fragmented DNA from euchromatin by three sequential treatments: 1. Acid, 2. Mild base, 3. Hot salt. The data indicate solubilization is effected by 1) depurination, 2) DNA denaturation, 3) chain breakage of the depurinated sites respectively in the three treatments. Conditions were found wherein each treatment in proper sequence was necessary for C-banding and the appropriate chemical reactions were measured in these treatment conditions. The acid treatment (0.2 N HCl) depurinates chromosomal DNA at the rate of 0.26×10^–6 purines/dalton min to an alkaline molecular weight of 10⁵ daltons but does not break the depurinated sites. Bleomycin can substitute for acid as a base removing agent. Sodium borohydride, by reducing the depurinated sugar's aldehyde thereby preventing chain breakage by the -elimination reaction, reversibly inhibits DNA-extraction. Chain breakage at the DNA's apurinic sites occurs not in the 2 min mild alkali treatment where the half-life for breakage is 26 min but in the 18 h hot salt treatment where the half-life for chain breakage is 1–2 h. Most of the DNA extraction occurs in the hot salt as 10⁵ dalton fragments as measured in formamide gradients. Bleomycin is introduced as a substitute for HCl; it removes nitrogenous bases from DNA in situ while better preserving the morphology of the final C-banded chromosomes.     

Recent Origin of Dioecious and Gynodioecious Y Chromosomes in Papaya

Sex of dioecious and gynodioecious papayas is controlled by two slightly different Y chromosomes, Y for males and Y^h for hermaphrodites. All combinations of the Y and/or Y^h chromosomes are lethal. We investigated the features of paired dioecious X- and Y-specific bacterial artificial chromosomes (BACs) and compared their sequences to corresponding gynodioecious X- and Y-specific BACs. Numerous chromosomal rearrangements were detected between the X- and Y-specific BACs, including inversions, deletions, insertions, and duplications. DNA sequence expansion was documented on the Y BAC. Dioecious and gynodioecious X-specific BACs were virtually identical. The Y- and Y^h-specific BACs shared high degree of DNA sequence identity, but local chromosomal rearrangements were detected, as the consequence of suppression of recombination in the male specific region and the isolation of Y and Y^h chromosomes enforced by the lethal effect. Analysis of sequence divergence between three dioecious X and Y gene pairs resulted in the estimated ages of divergence from 0.6 to 2.5 million years, reinforcing the hypothesis of a recent origin of the papaya sex chromosomes. The estimated age of divergence between Y and Y^h chromosomes was approximately 73,000 years for Gene 5. Our findings indicate that Y and Y^h chromosomes evolved from a common ancestral Y chromosome, possibly prior to the origin of agriculture. The existence of a hermaphrodite Y^h chromosome is less likely to have resulted from human selection as once suggested.     

The replicative organization of DNA in polytene chromosomes ofDrosophila hydei

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with³H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10^-4 pg for the largest bands inDrosophila hydei. From the results of H³-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.     

Isolation and partial characterization of replication intermediates in Chironomus polytene chromosomes

DNA replication was investigated in cells with polytene chromosomes. The cells were obtained from the salivary glands of the dipteran Chironomus tentans. Polytene chromosomes are characterized by a specific and constant band — interband structure formed by the lateral association of homologous chromatids side by side. — The salivary gland DNA was labelled by injection of radioactive precursor into the living animal, extracted with a neutral nondenaturing buffer at 25° C and finally characterized by agarose gel electrophoresis. Radioactive DNA pulse-labelled for 30–60 min was released from the polytene chromosomes during cell lysis in the form of double-stranded fragments. The fragments, which show a heterogeneous appearance in gel electrophoresis, are probably produced in the living cell by the joining of several Okazaki fragments. The release of the fragments from the polytene chromosome is prevented by lysis at 0° C instead of 25° C. The size of the double-stranded fragments range between 3.75–6×10⁶ D. Moreover, after a time-lag the fragments are joined together to produce a high-molecular weight DNA. The existence of these nascent DNA fragments is discussed in relation to an earlier proposal that each band in the polytene chromosome may function as a separate replication unit.     

Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G₁/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 M). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×10⁵/root and 1.4×10⁵/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.     

Satellite DNA sequences flank amplified DHFR domains in marker chromosomes of mouse fibrosarcoma cells

This study centers on marker chromosomes carrying expanded chromosomal regions which were observed in two independent derivatives of the AA12 murine fibrosarcoma line, the 10^–3 M MTX-res H2 and the 5×10^–7 M MTX-res E. Previous characterization of the marker chromosomes of MTX-res variants showed their common derivation from a marker chromosome (m) of the parental line, endowed with two interstitial C-bands. Cytogenetic evidence pointed to one C-band ofm as the site involved in the chromosomal rearrangements leading to the HSR/ASR chromosomes. ISH of a³H-labeled satellite DNA probe allowed satellite sequences flanking the HSR/ASR in the marker chromosomes, where the C-band was no longer visible, to be detected. FISH experiments using biotinylated DHFR and satellite DNA probes showed that the respective target sequences are contiguous in new marker chromosomes. They also allowed inter- and intrachromosomal rearrangements to be seen at DHFR amplicons and satellite sequences. Double-color FISH using digoxygenated satellite DNA and biotinylated pDHFR7 showed that in a marker chromosome from the H2 cell line the two target sequences are not only adjacent, but closer than 3 Mb, as indicated by overlapping of the different fluorescence signals given by the two probes. Another marker chromosome in the E variant was shown to display a mixed ladder structure consisting of a head-to-head tandem of irregularly-sized satellite DNA blocks, with two symmetrical interspersed DHFR clusters.Abbreviations DHFR dihydrofolate reductase - MTX Methotrexate - HSR Homogeneously Staining Region - ASR Abnormally Staining Region - DM Double Minute - ISH In Situ Hybridization - FISH FluorescenceIn Situ Hybridization     

MAMMALIAN CHROMOSOMES IN VITRO : XVIII. DNA Replication Sequence in the Chinese Hamster

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H³-thymidine labeling and the method of 5-fluorodeoxyuridine block with H³-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.     

Isolation and some properties of a mammalian ribosomal DNA

The DNA coding for 28 S and 18 S ribosomal RNA, including the spacer regions, has been isolated from calf (Bos taurus) thymus gland. The method used included shearing of the total DNA to a highly homogeneous size population, selective heat denaturation and S 1 nuclease treatment to remove single stranded DNA. Repeated centrifugation on density gradients yields a 140-fold purified rDNA fraction with a GC content of 61.2%. Eco RI nuclease cleaves this DNA into two fragments of 16.4 and 4.9×10⁶ daltons. Hybridization of these fragments with 28 S and 18 S rRNA shows that the 28 S coding sequence is located mostly on the 4.9×10⁶ dalton fragment, while both the 16.4 and 4.9×10⁶ dalton fragments contain the 18 S sequence. The data indicate that the ribosomal RNA gene has a repeat unit of 21.3×10⁶ daltons which includes a nontranscribed spacer of about 12.5×10⁶ daltons.     

Lifestyle factors and sperm aneuploidy

Different environmental and lifestyle factors may interfere with the normal disjunction of sister chromatids/chromosomes during meiosis and may cause aneuploidy. The aim of the study was to examine the association between lifestyle factors and sperm aneuploidy. The study population consisted of 212 healthy men under 45 years of age attending an infertility clinic for diagnostic purposes and who had a normal semen concentration of 20–300 × 10⁶ mL or slight oligozoospermia (semen concentration of 15–20 × 10⁶/mL). All participants were interviewed and provided a semen sample. Sperm aneuploidy was assessed using multicolor FISH (DNA probes specific for chromosomes X, Y, 18, 13, 21). Results from the study suggest that lifestyle factors are related to sperm aneuploidy. A positive relationship was found between coffee drinking everyday and the lack of chromosome X or Y, as well as coffee drinking 1–6 times per week and additional chromosome 18. Wearing boxer shorts decrease the copy number changes in the whole chromosome 18, the number of additional chromosome 18 and the lack of chromosome 13. Additionally, obesity (BMI 30–40 kg/m²) was positively associated with additional chromosome 21 after being adjusted for potential confounders. These findings demonstrate that changing the men's lifestyle habits may contribute to reduction of the incidence of sperm aneuploidy. It is necessary that men continue to follow sensible health advice concerning excess weight, coffee drinking and wearing tight fitting underwear. As this is the first such study to examine different lifestyle factors and sperm aneuploidy, the results need to be confirmed on larger population.