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1.
Subpopulations of rat lung fibroblasts with different amounts of type I and type III collagen mRNAs 总被引:3,自引:0,他引:3
A fluorescence-based method using the cell sorter has been devised to separate rat lung fibroblasts into subpopulations. Type I or type III collagen antiserum was used as the primary antibody to react with parent rat lung fibroblasts. This was followed by a fluorescein-conjugated secondary antibody. Specificity of the primary collagen antibody was determined using a monoclonal beta-actin antibody and purified IgG as the primary antibodies. The fluorescent shift of parent rat lung fibroblasts was optimized for the amount of primary collagen antibody and secondary fluorescein-conjugated antibody. An increase in slot blot intensity was observed for pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) mRNAs with increasing amounts of cellular RNA. When precipitating with type I collagen antibodies, the total cellular steady-state levels of type I procollagen mRNAs were increased in the high intensity cells as compared with the low intensity cells. Alternately, when the type III collagen antibodies were used to precipitate the rat lung fibroblasts, the low intensity cells had increased type I procollagen mRNAs while the high intensity cells had increased type III procollagen mRNA. The subpopulations of rat lung fibroblasts after isolation using the fluorescent cell sorter were readily propagated for at least four passages. 相似文献
2.
The alpha-like globin gene cluster in rabbits contains embryonic zeta-
globin genes, an adult alpha-globin gene, and theta-globin genes of
undetermined function. The basic arrangement of genes, deduced from
analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta
2-zeta 3-theta 2-3'. However, the pattern of restriction fragments
containing zeta- and theta-globin genes varies among individual rabbits.
Analysis of BamHI fragments of genomic DNA from 24 New Zealand white
rabbits revealed eight different patterns of fragments containing
zeta-globin genes. The large BamHI fragments containing genes zeta 0 and
zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the
zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary
in size. In contrast to this constancy in the size of the restriction
fragments, the copy number of the zeta 2 and zeta 3 genes does vary among
different rabbits. No length polymorphism was detected in the BamHI
fragments containing the theta-globin genes, but again the copy number
varies for restriction fragments containing the theta 2 gene. The alpha 1-
and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI
fragment. The combined data from hybridization with both zeta and theta
probes shows that the BamHI cleavage pattern does not vary within the
region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern
genomic blot-hybridization patterns for the progeny of parental rabbits
with different zeta-globin gene patterns shows that the polymorphic
patterns are inherited in a Mendelian fashion. Two different haplotypes
have been mapped based on the genomic blot-hybridization data. The
variation in the alpha-like globin gene cluster in the rabbit population
results both from differences in the copy number of the duplication block
containing the zeta-zeta-theta gene set and from the presence or absence of
polymorphic BamHI sites.
相似文献
3.
The Behavior of Insertions near a Site of Mitotic Gene Conversion in Yeast 总被引:3,自引:1,他引:2
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In yeast, coincident gene conversion events involving the LEU1 and TRP5 loci (16 cM apart) occur at frequencies that are far greater than is expected for two independent acts of recombination. When a large plasmid (pJM53) is placed between these genes so that a direct repeat is produced, there is frequent loss of the insert among coincident convertants. Previous results strongly suggest that this is due to a separate, intrachromosomal exchange between the direct repeats rather than to excision from an extensive region of heteroduplex DNA. In this paper, we extend our genetic and molecular analysis to a plasmid insertion (pKSH) which replaces rather than duplicates the chromosomal material. The relative stabilities of pKSH and pJM53 are compared among coincident Leu+Trp+ convertants and convertants involving only one locus (LEU1). The pKSH insertion is significantly more stable in the latter which constitute a large majority of the selectable recombinants. In the former, both insertions are lost with high frequency. These results are used to argue that, while most mitotic conversion does not result from long intermediates, coincident convertants may arise from either multiple intermediates or extensive heteroduplex regions. 相似文献
4.
Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution 总被引:1,自引:0,他引:1
In order to study the relationships among mammalian alpha-globin genes, we
have determined the sequence of the 3' flanking region of the human alpha 1
globin gene and have made pairwise comparisons between sequenced
alpha-globin genes. The flanking regions were examined in detail because
sequence matches in these regions could be interpreted with the least
complication from the gene duplications and conversions that have occurred
frequently in mammalian alpha-like globin gene clusters. We found good
matches between the flanking regions of human alpha 1 and rabbit alpha 1,
human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and
horse alpha 1 and goat II alpha. These matches were used to align the
alpha-globin genes in gene clusters from different mammals. This alignment
shows that genes at equivalent positions in the gene clusters of different
mammals can be functional or nonfunctional, depending on whether they
corrected against a functional alpha-globin gene in recent evolutionary
history. The number of alpha-globin genes (including pseudogenes) appears
to differ among species, although highly divergent pseudogenes may not have
been detected in all species examined. Although matching sequences could be
found in interspecies comparisons of the flanking regions of alpha- globin
genes, these matches are not as extensive as those found in the flanking
regions of mammalian beta-like globin genes. This observation suggests that
the noncoding sequences in the mammalian alpha-globin gene clusters are
evolving at a faster rate than those in the beta-like globin gene clusters.
The proposed faster rate of evolution fits with the poor conservation of
the genetic linkage map around alpha-globin gene clusters when compared to
that of the beta-like globin gene clusters. Analysis of the 3' flanking
regions of alpha-globin genes has revealed a conserved sequence
approximately 100-150 bp 3' to the polyadenylation site; this sequence may
be involved in the expression or regulation of alpha-globin genes.
相似文献
5.
A Rapid Chromosome-Mapping Method for Cloned Fragments of Yeast DNA 总被引:19,自引:2,他引:17
A rapid and generally applicable method is described for mapping a cloned yeast DNA segment to the chromosome(s) from which it originated. The method is based upon the recent finding that the integration into a yeast chromosome of a segment of the 2 mu plasmid DNA results, in heterozygous diploids, in the specific loss of genetic information from the chromosome into which the 2 mu DNA was integrated (Falco et al. 1982). After verification of the accuracy of the method using several genes whose position was known in advance, the method was used to locate the yeast actin gene, which lies on the left arm of chromosome VI, about 50 cM distal to CDC4. 相似文献
6.
Elena De Falco Gaia Scafetta Chiara Napoletano Rosa Puca Enzo Maria Vingolo Giuseppe Ragona Olga Iorio Giacomo Frati 《Cell and tissue banking》2013,14(2):277-287
Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm2 undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes. 相似文献
7.
Zhiying You Mariarosaria De Falco Katsuhiko Kamada Francesca M. Pisani Hisao Masai 《PloS one》2013,8(8)
The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes. 相似文献
8.
Competition regulation in taekwondo has experienced several changes during the last few years, for example, kicks to the head score more points than kicks to the chest. In addition, some external factors such as the height of target and execution distance seem to affect the kick performance. The aim of this study was to analyse selected biomechanical parameters (impact force, reaction time, and execution time) according to the height and execution distance in two different male groups (experts (n = 12) and novices (n = 21)). Athletes kicked twice from every execution distance (short, normal and long) and towards two different heights of target (chest and head) in a random order. Novices kicked to the head with a longer reaction time than to the chest (p < 0.05) but experts were able to kick with similar performance for both heights. From short and normal distances experts kicked with similar performance; whereas from the normal distance novices had longer reaction and execution time than from the short distance (p < 0.05). In conclusion, in counterattacking situations, experts should perform the roundhouse kick to the head instead of to the chest, because it produces better scores with similar performance; whereas novice athletes should avoid kicking to the head because they are not able to kick with similar performance. Moreover, it is recommended that during counterattacks higher-level taekwondo athletes should intend to kick from normal distances. 相似文献
9.
Daniela Tagliaferri Maria Teresa De Angelis Nicola Antonino Russo Maria Marotta Michele Ceccarelli Luigi Del Vecchio Mario De Felice Geppino Falco 《PloS one》2016,11(2)
Pluripotency confers Embryonic Stem Cells (ESCs) the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. Although the majority of ESCs divide without losing pluripotency, it has become evident that ESCs culture consists of multiple cell populations with different degrees of potency that are spontaneously induced in regular ESC culture conditions. Zscan4, a key pluripotency factor, marks ESC subpopulation that is referred to as high-level of pluripotency metastate. Here, we report that in ESC cultures treated with retinoic acid (RA), Zscan4 ESCs metastate is strongly enhanced. In particular, we found that induction of Zscan4 metastate is mediated via RA receptors (RAR-alpha, RAR-beta, and RAR-gamma), and it is dependent on phosphoinositide-3-kinase (PI3K) signaling. Remarkably, Zscan4 metastate induced by RA lacks canonical pluripotency genes Oct3/4 and Nanog but retained both self-renewal and pluripotency capabilities. Finally we demonstrated that the conditional ablation of Zscan4 subpopulation is dispensable for both endoderm and mesoderm but is required for ectoderm lineage. In conclusion, our research provides new insights about the role of RA signaling during ESCs high pluripotency metastate fluctuation. 相似文献
10.
Grauso Laura Emrick Stefano de Falco Bruna Lanzotti Virginia Bonanomi Giuliano 《Phytochemistry Reviews》2019,18(4):1115-1132
Phytochemistry Reviews - Phytoalimurgy is a term that derives from Greek and Latin by combination of the words φυτόν, which meaning plant, and alimenta urgentia, indicating... 相似文献