Engineering mammalian chromosomes

Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic alpha-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homo- geneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that alpha-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.     

The terminal DNA structure of mammalian chromosomes

In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated.     

Two-parameter flow fluorescence analysis of human chromosomes. Quantitative processing]

Adrenomedullin (AM) is a newly discovered hypotensive peptide which is believed to play an important role for blood pressure control in the adult. Although it has been well established that a major production site of AM is vascular endothelial cells, we now show that AM is most highly expressed in trophoblast giant cells, which are derived from the conceptus and are directly in contact with maternal tissues at the implantation site. Northern blot and in situ hybridization analyses show that the AM mRNA begins to be detected just after implantation and its level peaks at 9.5 days postconception (d.p.c.) in those cells. Expression then falls dramatically after 10.5 d.p.c., coincident with the completion of the mature chorioallantoic placenta. Immunohistochemical analyses show that the AM peptide is secreted from the trophoblast giant cells into the surrounding tissues, i.e., embryo, decidua, and maternal circulation. In contrast, the expression of an AM receptor was not detected by Northern blot analyses in either embryo or trophoblast giant cells at 7 d.p.c., when the AM gene is most highly expressed in the trophoblast giant cells. This suggests that the AM produced and secreted from the embryo's trophoblast giant cells acts on the maternal tissues rather than on the embryonic tissues. Based on these results, we propose that the high production of AM may be the mechanism by which the embryos survive at the early postimplantation period by pooling maternal blood in the implantation site in order to secure nutrition and oxygen before the establishment of efficient embryo-maternal circulation through the mature placenta.     

FISH, interphase cytogenetics, gene rearrangements]

OBJECTIVE: To investigate the possible involvement of chromosome abnormalities in pathogenesis of human esophageal cancer. METHODS: Four cell lines of human esophageal cancer (EC) established in our laboratory were analysed using interphase fluorescence in situ hybridization (FISH), chromosome painting technique and comparative genomic hybridization (CGH). RESULTS: Chromosome gain of 1,2,3,8,16, 17, and 20 was found in the four cell lines, and loss of chromosome Y in cell line EC8712, EC8733 and EC8501 was noted. Other frequent changes were partial deletion of 1p, translocation of 2q and amplification of 5p in all 4 cell lines, and amplification of 8q and 13q in EC8733 and deletion of 17p in EC8712. CONCLUSION: The data suggest that nonrandom chromosome aberrations may play an important role in the pathogenesis of human esophageal cancer.     

Molecular analysis of mammalian timeless

We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.     

Comparative analysis of mammalian stanniocalcin genes

The recent discovery of mammalian stanniocalcin (STC) prompted an investigation of its gene structure and expression pattern to study its function and regulation. We show that both the human and mouse genes are composed of four exons spanning about 13 kb, with 85% nucleotide sequence identity in coding regions. Remarkably high sequence conservation between species also exists in the approximately 3-kb 3'-untranslated region. Comparative analysis of the 5'-untranslated region and flanking DNA from the rat and human STC genes showed long stretches of CAG trinucleotide repeats and an additional (CA)25 dinucleotide repeat unique to the rat promoter. An analysis of STC expression in the mouse showed that ovary contained the highest level of messenger RNA, with lower, but detectable, levels in most tissues. In situ hybridization revealed strong, specific hybridization over the thecal-interstitial cells of the ovarian stroma, whereas immunohistochemical analysis indicated that STC was present not only in the stroma, but also in the corpora lutea and oocyte of the developing follicle. Consequently, STC may act as a signaling molecule between the thecal-interstitial cell compartment and the corpus luteum and oocyte, thereby regulating the activity of these structures in some way. These findings suggest that in addition to its role in mineral metabolism, STC has acquired an important function in reproduction during its evolution to mammals.     

An important subgroup of phyllodes tumors of the breast is characterized by rearrangements of chromosomes 1q and 10q

From a study of 10 cases of our own and 13 cases of the literature, anomalies of chromosome 1q and 10q emerge as consistently occurring changes in an important subgroup of phyllodes tumors of the breast. Anomalies of chromosome 1 were the most frequent ones, with a gain of 1q material, and histologically the tumors in which these anomalies were found were low grade malignancies. Structural changes of 10q emerged as the second most frequent chromosome change.     

Computer-assisted analysis of chromosomes of the European crested newt

A computer programme has been developed for partly automating the process of chromosome analysis of the European crested newt (Triturus cristatus carnifex). Chromosome classifications made by the computer and by the cytologist have been compared for a sample of 130 spermatogonial metaphase. The twelve pairs of chromosomes have been classified into three groups. The overall error rate achieved by the computer was 14% for chromosome assignment to single pairs, and 3.8% for chromosome assignment to the proper group.     

热态铸余渣返生产可行性分析

铸余渣综合利用是钢铁企业节能减排的重要方法.在分析铸余渣资源属性的基础上,结合炼钢工艺流程,研究了铸余渣返生产的工艺路径和关键技术,重点讨论了返生产过程中由于温降而产生的粘罐和铸余渣与铁水混兑时的喷爆问题,提出了通过调整钢水碳含量解决钢水粘罐问题的技术措施和通过实验室研究解决喷爆的思路.同时结合现场生产管理调度,分析了铸余渣返生产后的炼钢工艺优化方向,为铸余渣的资源化利用提供了依据.     

球磨机反转可行性分析

我厂现用的球磨机都是一个方向旋转。当大齿轮单面使用周期到后,需要翻面,这就需要停磨3～4天,且检修工作量也大。若使球磨机反转,就可以直接使用大齿轮的另一面。本文通过对小轴承座.大瓦等部位的受力分析,从理论方面对球磨机的反转可行性进行了分析。     

联合转炉放散炉气预热废钢的可行性分析

研究了转炉放散炉气在竖炉中预热废钢的传热规律,建立了废钢多孔介质中的瞬态非热平衡流固耦合模型;通过FLUENT模拟得出废钢传热特性曲线和废钢料层内部的温度分布,分析了废钢预热中料层高度、转炉放散炉气温度和进气速度对废钢温度及预热效率的影响.研究结果表明:在1 200℃炉气中,废钢经30 min预热后温度达到570.3℃,预热效率为38.3%;随着废钢料层高度从2.5 m降低到0.7 m,废钢温度从570.3℃升高到695.7℃,预热效率下降为13.3%;随着转炉放散炉气温度从1 200℃升高到1 600℃,废钢温度从570.3℃升高到734.4℃,预热效率上升为44.2%;随着进气速度从2.375 m/s降低到1.125 m/s,废钢温度从570.3℃下降到356.1℃,预热效率上升为54.5%.     

包钢开发生产轴承钢的可行性分析

介绍轴承钢的种类及应用 ,轴承钢的冶炼、浇注工艺 ,夹杂物对轴承钢性能的影响等 ,分析包钢在开发生产轴承钢方面主要存在的优势、问题及前景     

宣钢取消混铁炉可行性分析

在对宣钢转炉炼钢厂的生产情况进行调研的基础上,对炼钢厂混铁炉的存在提出了置疑,并借鉴沙钢的"铁水包多功能"模式,提出取消混铁炉的可行性措施.     

A proposed path by which genes common to mammalian X and Y chromosomes evolve to become X inactivated

Mammalian X and Y chromosomes evolved from an autosomal pair; the X retained and the Y gradually lost most ancestral genes. In females, one X chromosome is silenced by X inactivation, a process that is often assumed to have evolved on a broadly regional or chromosomal basis. Here we propose that genes or clusters common to both the X and Y chromosomes (X-Y genes) evolved independently along a multistep path, eventually acquiring dosage compensation on the X chromosome. Three genes studied here, and other extant genes, appear to be intermediates. ZFX, RPS4X and SMCX were monitored for X inactivation in diverse species by assaying CpG-island methylation, which mirrors X inactivation in many eutherians. ZFX evidently escaped X inactivation in proto-eutherians, which also possessed a very similar Y-linked gene; both characteristics were retained in most extant orders, but not in myomorph rodents. For RPS4X, escape from X inactivation seems unique to primates. SMCX escapes inactivation in primates and myomorphs but not in several other lineages. Thus, X inactivation can evolve independently for each of these genes. We propose that it is an adaptation to the decay of a homologous, Y-linked gene.     

Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes

For determination of the extent to which ribosomal DNA (rDNA) is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes. A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed. In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert. By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes. Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines. When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments. If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.     

兰炭作为酒钢高炉喷吹用煤的可行性分析

通过兰炭和酒钢高炉喷吹用煤的工业分析和燃烧性能对比分析,采用新疆兰炭作为高炉喷吹原料是可行的。     

南钢电炉厂连铸提高拉速的可行性分析

通过对提高连铸拉速的原因及其铸坯质量分析，总结出本连铸机提高拉速的可行性及提高拉速过程中为保证质量所规定的限制条件。     

Excision of 7-bromomethylbenz[a]anthracene--DNA adducts in replicating mammalian cells

The excision of 7-bromomethylbenz[a]anthracene--DNA adducts was studied in two cell lines (HeLa S-3 and Chinese hamster V-79379A). In both cell lines, carcinogen-modified adenine residues were excised more readily than the modified guanine residues and the percentage of the total products excised decreased after treatment with higher concentrations of carcinogen. At the highest concentrations used in the Chinese hamster cells, neither DNA synthesis nor excision was detected. The lowest concentration used for these cells permitted almost 100% survival and all the DNA was replicated in a 30-h interval even though 50% of the initial damage was still present. The two- to threefold lower sensitivity of the Chinese hamster cells (compared with the Hela cells) to the carcinogen is attributed to this capacity for replication of DNA on a damaged template since the two cell lines' capacities for excision of the chemical damage were found to be comparable.     

A proposed new mammalian cell protein kinase, associated with microtubules]

The effect of phospholipase A2 treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with Naja mocambique mocambique phospholipase A2 reduced the pool sizes of phosphatidylcholine and phosphatidylethanolamine compared with controls. The pool sizes of lysophosphatidylcholine and lysophosphatidylethanolamine were elevated, whereas the pool sizes of cardiolipin and other phospholipids were unaffected by phospholipase A2 treatment. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or preincubated in the absence or presence of phospholipase A2. In all experiments, radioactivity incorporated into cardiolipin was reduced in phospholipase A2-treated cells with time compared with controls, indicating attenuated de novo biosynthesis of cardiolipin. The mechanism for the reduction in cardiolipin biosynthesis in phospholipase A2-treated cells was a decrease in the activity of phosphatidic acid:cytidine-5'-triphosphate cytidylyltransferase, the rate-limiting enzyme of cardiolipin biosynthesis, mediated by elevated cellular lysophosphatidylcholine levels. The results suggest that de novo cardiolipin biosynthesis in H9c2 cells may be regulated by the cellular level of lysophosphatidylcholine.