Chromosome segregation in crane-fly spermatocytes: cold treatment and cold recovery induce anaphase lag

Anaphase lagging of autosomes was observed in 6.1±5.4% of the primary spermatocytes in untreated larvae of the crane fly, Nephrotoma suturalis. Lagging was induced by exposure of larvae to 6 ° C and during recovery at 22 ° C from exposure to 0.2, 2, and 6 ° C. The incidence of anaphase lag was maximal at 80 to 90 min of recovery. Induced lagging was observed at that recovery time after exposures of only 2.5 h to 2 or 0.2 ° C, but its incidence increased with longer exposures. As many as 85% of the cells in anaphase contained autosomal laggards after 61 h at 2 ° C and 80 to 90 min of recovery. At 2 ° C, cells reached the prophase-prometaphase transition, but spindles did not appear to form. Those cells proceeded through prometaphase during recovery, reaching mid-anaphase after 80 to 90 min of recovery. Chromosomes that lagged at anaphase during recovery from 2 ° C were observed in living cells to be half-bivalents derived from bivalents that congressed to the metaphase plate. One or both half-bivalents of any bivalent could lag. In some cells, one half-spindle had more half-bivalents than the other. Cells with autosomal laggards often did not cleave, and in uncleaved cells the second division employed spindles having two, three, or four poles. The basis of induced lagging might be a lapse in spindle attachment or motive force application at the start of anaphase or a failure of chromosomes to achieve proper orientation before the onset of anaphase.     

Malorientation in half-bivalents at anaphase: analysis of autosomal laggards in untreated, cold-treated, and cold-recovering crane fly spermatocytes

Exposing crane fly larvae to 6 degrees C or returning them to 22 degrees C after exposure to 6, 2, or 0.2 degrees C can induce any number of autosomes in their primary spermatocytes to lag near the spindle equator at anaphase. Autosomal laggards in cold-recovering cells are contained in bivalents until anaphase (Janicke, M. A., and J. R. LaFountain, 1982, Chromosoma, 85:619-631). We report here documentation that lagging autosomes in cold-treated and cold- recovering cells are maloriented. During meiosis I, half-bivalents usually associate with only one pole via kinetochore fibers, with sister chromatids being oriented to the same pole. In contrast, laggards had kinetochore microtubules (kMTs) extending from them toward both poles: one sister was oriented to one pole and the other had some or all of its kMTs extending toward the opposite pole. Bipolar malorientation of autosomal laggards also was observed in one untreated cell. The number of kMTs per half-bivalent was similar in lagging and non-lagging autosomes, and those kMTs were contained in long birefringent kinetochore fibers. The overall spindle structure in cold- recovering cells was similar to that observed in untreated anaphase cells. Giemsa-stained centromeric dots of sister chromatids were contiguous in non-laggards and separated in laggards at anaphase. We conclude that bipolar malorientations can exist at anaphase in chromosomes that remain paired until anaphase, that cold recovery increases the frequency of that anomaly, and that such malorientations may be one cause of anaphase lag.     

Chromosome elimination in micronuclei: a common cause of hypoploidy.

An excess of hypoploid cells has repeatedly been reported in studies of aneuploidy and has often been attributed to technical artifact. We have examined at least 200 anaphase or early-telophase cells from each of 28 normal women and found that chromosome or chromatid lagging occurs in an average of 2.43% of cells. In a separate study, we have examined the frequency of micronuclei in cytochalasin B-arrested, binucleate cells and shown that a similar frequency of cells (1.6%) contain one or more micronuclei. Using in situ hybridization of an alpha centromeric probe (alpha R1), which hybridizes to 9 of the 22 human autosomes, we were able to infer that most, if not all, of the micronuclei contain whole chromosomes or chromatids. Since the loss of a chromosome by lagging will induce hypoploid daughter nuclei (two where a chromosome is lost and one where a chromatid is lost), we conclude that lagging is a major mechanism for chromosome loss in human lymphocyte cultures. This loss occurs in the cells of normal individuals under control conditions.     

First evidence of sex chromosome pre-reduction in male meiosis in the Miridae bugs (Heteroptera)

The karyotype and male meiosis of Macrolophus costalis Fieber (Insecta, Heteroptera, Miridae) were studied using C-banding, AgNOR-banding and DNA sequence specific fluorochrome staining. The chromosome formula of the species is 2n = 28(24+X1X2X3Y). Male meiotic prophase is characterized by a prominent condensation stage. At this stage, two sex chromosomes, "X" and Y are positively heteropycnotic and always appeared together, while in autosomal bivalents homologous chromosomes were aligned side by side along their entire length, that is, meiosis is achiasmatic. At metaphase I, "X" and Y form a pseudobivalent and orient to the opposite poles. At early anaphase I, the "X" chromosome disintegrates into three separate small chromosomes, X1, X2, and X3. Hence both the autosomes and sex chromosomes segregate reductionally in the first anaphase, and separate equationally in the second anaphase. This is the first evidence of sex chromosome pre-reduction in the family Miridae. Data on C-heterochromatin distribution and its composition in the chromosomes of this species are discussed.     

Age-dependent inclusion of sex chromosomes in lymphocyte micronuclei of man.

Two-color centromeric FISH was used to study the inclusion of the X and Y chromosomes in micronuclei of cultured lymphocytes from 10 men representing two age groups (21-29 years and 51-55 years). In addition, pancentromeric FISH was separately performed to identify any human chromosomes in micronuclei. One hundred micronuclei per probe were examined from each donor. A higher mean frequency of Y-positive micronuclei was observed in the older men than in the younger men. In both age groups, the X chromosome was micronucleated clearly more often than expected by chance, and the Y chromosome was overrepresented in micronuclei among the older men but not among the younger men. In lymphocytes of four women, X-positive micronuclei were more frequent than they were in men, even after the fact that women have two X chromosomes was taken into account. Similar results were obtained in first-division lymphocytes identified by cytochalasin-B-induced cytokinesis block. In comparison with normal cells, these binucleate cells showed a higher frequency (per 1,000 nuclei) of X-positive micronuclei (in the older men) but a lower frequency of micronuclei harboring autosomes or acentric fragments. In conclusion, the results show that both the X chromosome and the Y chromosome are preferentially micronucleated in male lymphocytes, the Y chromosome only in older subjects. Although the X chromosome has a general tendency to be included in micronuclei, it is micronucleated much more often in women than in men, which is probably the main reason for the high micronucleus frequency in women that has been documented in many previous studies.     

Orientation of nonrandomly segregating sex chromosomes in spermatocytes of the flea beetle, Alagoasa bicolor L.

In males of the flea beetle, Alagoasa bicolor L., spermatocytes have two achiasmate sex chromosomes, X and Y, each of which is approximately five times larger than the ten pairs of chiasmate autosomes. At metaphase I, these univalent sex chromosomes are located on a spindle domain separated from the autosomal spindle domain by a sheath of mitochondria. A single centriole pair is located at each pole of the spindle. In prometaphase I, each sex chromosome appears to maintain an attachment to both spindle poles via kinetochore microtubules (i.e., amphitelic orientation). Before anaphase I, this orientation changes to the syntelic orientation (both sister kinetochores connected to the same pole), perhaps by the release of microtubule attachments from the more distant pole by each of the chromosomes. The syntelic orientation just prior to anaphase I leaves each sex chromosome attached to the nearest pole via kinetochore microtubules, ensuring nonrandom segregation. As the sex chromosomes reorient, the autosomes follow in a sequential manner, starting with the bivalent closest to the sex spindle domain. We report here data that shed new light on the mechanism of this exceptional meiotic chromosome behavior.     

Adaptive protein evolution of X-linked and autosomal genes in Drosophila: implications for faster-X hypotheses

Patterns of sex chromosome and autosome evolution can be used to elucidate the underlying genetic basis of adaptative change. Evolutionary theory predicts that X-linked genes will adapt more rapidly than autosomes if adaptation is limited by the availability of beneficial mutations and if such mutations are recessive. In Drosophila, rates of molecular divergence between species appear to be equivalent between autosomes and the X chromosome. However, molecular divergence contrasts are difficult to interpret because they reflect a composite of adaptive and nonadaptive substitutions between species. Predictions based on faster-X theory also assume that selection is equally effective on the X and autosomes; this might not be true because the effective population sizes of X-linked and autosomal genes systematically differ. Here, population genetic and divergence data from Drosophila melanogaster, Drosophila simulans, and Drosophila yakuba are used to estimate the proportion of adaptive amino acid substitutions occurring in the D. melanogaster lineage. After gene composition and effective population size differences between chromosomes are controlled, X-linked and autosomal genes are shown to have equivalent rates of adaptive divergence with approximately 30% of amino acid substitutions driven by positive selection. The results suggest that adaptation is either unconstrained by a lack of beneficial genetic variation or that beneficial mutations are not recessive and are thus highly visible to natural selection whether on sex chromosomes or on autosomes.     

Isolation and characterization of cloned human DNA fragments carrying reiterated sequences common to both autosomes and the X chromosome.

Several recombinants were identified and purified from a cloned library of human DNA by virtue of their homology to DNA from a mouse-human hybrid cell line containing a single human chromosome, the X, and their lack of homology to mouse DNA. Three recombinants were characterized in detail, and all were homologous to reiterated DNA from the human X chromosome. These recombinants also were homologous to reiterated sequences on one or more human autosomes and, therefore, were not X chromosome specific. The recombinant DNA fragments homologous to human reiterated X DNA were the same fragments homologous to human reiterated autosomal DNA. Digestion of genomic DNAs with several restriction enzymes revealed that the pattern of fragments homologous to one recombinant, lambda Hb2, was the same on autosomes as on the X chromosome, suggesting that the molecular organization of these elements on the X is not distinct from their organization on autosomes.     

Malorientation in half-bivalents at anaphase in crane fly spermatocytes following Colcemid treatment

Addition of Colcemid to the medium in which larvae of the crane fly Nephrotoma suturalis are cultivated induces a number of anomalous patterns of chromosome segregation. One of these is the anaphase lagging of autosomal half-bivalents. To investigate the cause of anaphase lagging, the orientation of sister kinetochores in Colcemidtreated spermatocytes having lagging half-bivalents was analyzed in serial sections. In contrast to nonlaggard halfbivalents that had pure syntelic orientation (sister kinetochores having all of their kinetochores microtubules (KMTs) extending to the same pole), six of the seven autosomal laggards that were selected for analysis had kinetochores with either amphitelic orientation (sister kinetochores each with a bundle of KMTs extending to opposite poles) or merotelic orientation (a single kinetochore having KMTs extending toward both poles). An additional laggard had syntelic orientation but two of the microtubules that were in its kinetochore fiber passed through the kinetochore and extended beyond it toward the equator. The bipolar malorientations observed in anaphase half-bivalents are interpreted to be a cause of the anaphase lagging induced by Colcemid treatment. Furthermore, it is hypothesized that such bipolar malorientations also may be stabilized at metaphase and thus explain the unusual tilting of metaphase bivalents commonly observed in Colcemid-treated cells.     

Elevated male European and female African contributions to the genomes of African American individuals

The differential relative contribution of males and females from Africa and Europe to individual African American genomes is relevant to mapping genes utilizing admixture analysis. The assessment of ancestral population contributions to the four types of genomic DNA (autosomes, X and Y chromosomes, and mitochondrial) with their differing modes of inheritance is most easily addressed in males. A thorough evaluation of 93 African American males for 2,018 autosomal single nucleotide polymorphic (SNP) markers, 121 X chromosome SNPs, 10 Y chromosome haplogroups specified by SNPs, and six haplogroup defining mtDNA SNPs is presented. A distinct lack of correlation observed between the X chromosome and the autosomal admixture fractions supports separate treatment of these chromosomes in admixture-based gene mapping applications. The European genetic contributions were highest (and African lowest) for the Y chromosome (28.46%), followed by the autosomes (19.99%), then the X chromosome (12.11%), and the mtDNA (8.51%). The relative order of admixture fractions in the genomic compartments validates previous studies that suggested sex-biased gene flow with elevated European male and African female contributions. There is a threefold higher European male contribution compared with European females (Y chromosome vs. mtDNA) to the genomes of African American individuals meaning that admixture-based gene discovery will have the most power for the autosomes and will be more limited for X chromosome analysis. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Homologies between X and Y chromosomes detected by DNA probes: localisation and evolution.

We have isolated and characterized DNA probes that detect homologies between the X and Y chromosomes. Clone St25 is derived from the q13-q22 region of the X chromosome and recognizes a 98% homologous sequence on the Y chromosome. Y specific fragments were present in DNAs from 5 Yq-individuals and from 4 out of 7 XX males analysed. An X linked TaqI RFLP is detected with the St25 probe (33% heterozygosity) which should allow one to establish a linkage map including other polymorphic X-Y homologous sequences in this region and to compare it to a Y chromosome deletion map. Probe DXS31 located in Xp223-pter detects a 80% homologous sequence in the Y chromosome. The latter can be assigned to Yq11-qter outside the region which contains the Y specific satellite sequences. ACT1 and ACT2, the actin sequences present on the X and Y chromosomes respectively, have been cloned. No homology was detected between the X and Y derived fragments outside from the actin sequence. ACT2 and the Y specific sequence corresponding to DXS31 segregate together in a panel of Y chromosomes aberrations, and might be useful markers for the region important for spermatogenesis in Yq. Various primate species were analysed for the presence of sequences homologous to the three probes. Sequences detected by St25 and DXS31 are found only on the X chromosome in cercopithecoidae. The sequences which flank ACT2 detect in the same species autosomal fragments but no male specific fragments. It is suggested that the Y chromosome acquired genetic material from the X chromosome and from autosomes at various times during primate evolution.     

鸭跖草科杜若(Pollia japonica Thunb.)的减数分裂研究

首次对鸭跖草科杜若(Pollia japonicaThunb.)进行了花粉母细胞减数分裂观察,并重新报道了该种的染色体数目为2n=32。结果显示,减数分裂中期I构型为16Ⅱ,并且观察到次级联会现象。减数分裂后期I和后期Ⅱ存在落后染色体、染色体断片、二次分裂不同步等异常现象,统计各时期畸形率都低于10%。随机统计花粉粒活性,成熟率达到90%以上。这说明杜若的减数分裂过程基本正常,也证明了2n=32的体细胞染色体数目是可信的。     

Segregation of the amphitelically attached univalent X chromosome in the spittlebug <Emphasis Type="Italic">Philaenus spumarius</Emphasis>

In meiosis I, homologous chromosomes combine to form bivalents, which align on the metaphase plate. Homologous chromosomes then separate in anaphase I. Univalent sex chromosomes, on the other hand, are unable to segregate in the same way as homologous chromosomes of bivalents due to their lack of a homologous pairing partner in meiosis I. Here, we studied univalent segregation in a Hemipteran insect: the spittlebug Philaenus spumarius. We determined the chromosome number and sex determination mechanism in our population of P. spumarius and showed that, in male meiosis I, there is a univalent X chromosome. We discovered that the univalent X chromosome in primary spermatocytes forms an amphitelic attachment to the spindle and aligns on the metaphase plate with the autosomes. Interestingly, the X chromosome remains at spindle midzone long after the autosomes have separated. In late anaphase I, the X chromosome initiates movement towards one spindle pole. This movement appears to be correlated with a loss of microtubule connections between the kinetochore of one chromatid and its associated spindle pole.     

Nonrandom chromosome segregation in Neocurtilla (Gryllotalpa) hexadactyla: an ultrastructural study

During meiosis I in males of the mole cricket Neocurtilla (Gryllotalpa) hexadactyla, the univalent X1 chromosome and the heteromorphic X2Y chromosome pair segregate nonrandomly; the X1 and X2 chromosomes move to the same pole in anaphase. By means of ultrastructural analysis of serial sections of cells in several stages of meiosis I, metaphase of meiosis II, and mitosis, we found that the kinetochore region of two of the three nonrandomly segregating chromosomes differ from autosomal kinetochores only during meiosis I. The distinction is most pronounced at metaphase I when massive aggregates of electron-dense substance mark the kinetochores of X1 and Y chromosomes. The lateral position of the kinetochores of X1 and Y chromosomes and the association of these chromosomes with microtubules running toward both poles are also characteristic of meiosis I and further distinguish X1 and Y from the autosomes. Nonrandomly segregating chromosomes are typically positioned within the spindle so that the kinetochoric sides of the X2Y pair and the X1 chromosome are both turned toward the same interpolar spindle axis. This spatial relationship may be a result of a linkage of X1 and Y chromosomes lying in opposite half spindles via a small bundle of microtubules that runs between their unusual kinetochores. Thus, nonrandom segregation in Neocurtilla hexadactyla involves a unique modification at the kinetochores of particular chromosomes, which presumably affects the manner in which these chromosomes are integrated within the spindle.     

Meiotic chromosome structure. Kinetochores and chromatid cores in standard and B chromosomes of Arcyptera fusca (Orthoptera) revealed by silver staining.

The behaviour of two chromosome structures in silver-stained chromosomes was analyzed through the first meiotic division in spermatocytes of the acridoid species Arcyptera fusca. Results showed that at diakinesis kinetochores and chromatid cores are individualized while they associate in bivalents of metaphase I; only kinetochores and distal core spots associate in the sex chromosome. Metaphase I is characterized by morphological and localization changes of both kinetochores and cores which define the onset of anaphase I. These changes analyzed in both autosomes and in the sex chromosome allow us to distinguish among three different substages in metaphase I spermatocytes. B chromosomes may be present as univalents, bivalents, or trivalents. Metaphase I B univalents are characterized by separated cores except at their distal ends and individualized and flat sister kinetochores. At anaphase I sister kinetochores of lagging B chromatids remain connected through a silver-stained strand. The behaviour of cores and kinetochores of B bivalents is identical with that found in the autosomal bivalents. The differences in the morphology of kinetochores of every chromosome shown by B trivalents at metaphase I may be related to the balanced forces acting on the multivalent. The results show dramatic changes in chromosome organization of bivalents during metaphase I. These changes suggest that chromatid cores are not involved in the maintenance of bivalents. Moreover, the changes in morphology of kinetochores are independent of the stage of meiosis but correlate with the kind of division (amphitelic-syntelic) that chromosomes undergo.     

Meiotic studies in mice carrying the sex reversal (Sxr) factor

A sex reversal factor (Sxr) that causes mice having apparently normal X chromosomes to become phenotypically male is transmitted in an autosomal pattern. The origin of the Sxr factor is still unknown. It seems most likely that it has originated from an autosomal gene mutation or is the result of a translocation of part of the Y chromosome to one of the autosomes. Chromosomes from four XY and six XO mice carrying this sex reversal factor were examined in the diakinesis stage of meiosis. The following unusual observations were noted: (1) in XY males carrying the Sxr factor, the X and Y chromosomes were separated more often than in controls. (2) The Y chromosome tends to be closer to an autosome when the X and Y are separate than when the X and Y are attached. (3) A chromosome fragment was present in 4/226 cells from two XO males and a single cell from an XY, Sxr carrier. Although there is no direct evidence, these observations seem to favor the possibility that the Sxr factor involves a chromosomal rearrangement rather than a single gene mutation.     

X染色体的DNA序列结构不同于6、7、8、10、11、12号染色体

雌性哺乳动物X染色体上的大部分基因均因X染色体失活作用而失去表达能力 ,X染色体长臂表现失活更明显。虽然对X染色体失活的许多方面都有所了解 ,但是仍然不清楚失活信号沿着X染色体全长扩散的机制。为了了解X染色体是否有不同于其他染色体的基因组学特征 ,这些特征是否关系到X染色体的失活扩散和维持 ,分析 6、7、8、1 0、1 1、1 2号染色体和X染色体DNA序列 7碱基 (7nt)组合水平的结构是否显示差异。从NCBI基因库(http :∥www .ncbi.nlm .nih .gov genome guide)下载 7条染色体长臂各 6 0Mb区域。将这 6 0Mb区域分为 0 5Mb (或 5 0kb)一段 ,对每一段DNA做 7nt字符串组合分析 ,如 1～ 7,2～ 8,3～ 9…… ,记录每种 7nt字符串的频率 ,A、C、G和T4个硷基的 7nt字符串共有 4 7=1 6 384种组合。根据数字差异显示的结果 (http :∥www .ncbi.nlm .nih .gov genome guide) ,选择在扁桃腺生发中心B细胞中高表达的基因 70个 ,用以计算所有内含子 (有义链 )的 7nt频率值。每个内含子被记录为一组 7nt频率值 ,求和相同基因中的所有内含子相同 7nt字符串的频率值 ,再用该和乘以该基因的表达频率得该基因 7nt字符串的频率值 ,求和 70个基因的 7nt字符串的频率值称做intron 7nt,该值试图模拟细胞中RNA小片段的总和。     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

Partner choice in heterologous chromosome segregation of the Y chromosome in competitive situations in the oocyte of Drosophila melanogaster

Heterologous segregation of the Y chromosome and secondary non-disjunction of the X chromosomes in female meiosis of Drosophila melanogaster was investigated in ten different crosses where different constellations of translocation/inversion or translocation/translocation systems of the large autosomes were present in the female parent. It appeared that the Y chromosome always segregates from the shortest of the possible heterologous pairing partners. This may be due to size-dependent mechanism of so-called distributive disjunction or to the possibility that the shorter the chromosome element is, the more easily it moves in the nucleus of the oocyte. Secondary non-disjunction of the X chromosomes appeared to be lower the more possible autosomal pairing partners the Y chromosome had, suggesting that the autosomes effectively compete with the X chromosomes for pairing with the Y chromosome. An alternative explanation is that, due to interchromosomal effect on recombination, crossing over in the X chromosomes was different in different experiments.     

The influence of the Robertsonian translocation Rb(X.2)2Ad on anaphase I non-disjunction in male laboratory mice

A Robertsonian translocation in the mouse between the X chromosome and chromosome 2 is described. The male and female carriers of the Rb(X.2)2Ad were fertile. A homozygous/hemizygous line was maintained. The influence of the X-autosomal Robertsonian translocation on anaphase I non-disjunction in male mice was studied by chromosome counts in cells at metaphase II of meiosis and by assessment of aneuploid progeny. The results conclusively show that the inclusion of Rb2Ad in the male genome induces non-disjunction at the first meoitic division. In second metaphase cells the frequency of sex-chromosomal aneuploidy was 10.8%, and secondary spermatocytes containing two or no sex chromosome were equally frequent. The Rb2Ad males sired 3.9% sex-chromosome aneuploid progeny. The difference in aneuploidy frequencies in the germ cells and among the progeny suggests that the viability of XO and XXY individuals is reduced. The pairing configurations of chromosomes 2, Rb2Ad and Y were studied during meiotic prophase by light and electron microscopy. Trivalent pairing was seen in all well spread nuclei. Complete pairing of the acrocentric autosome 2 with the corresponding segment of the Rb2Ad chromosome was only seen in 3.2% of the cells analysed in the electron microscope. The pairing between the X and Y chromosome in the Rb2Ad males corresponded to that in males with normal karyotype. Reasons for sex-chromosomal non-disjunction despite the normal pairing pattern between the sex chromosomes may be seen in the terminal chiasma location coupled with the asynchronous separation of the sex chromosomes and the autosomes.(ABSTRACT TRUNCATED AT 250 WORDS)