A new site-specific endonuclease StuI from Streptomyces tubercidicus

A new sequence-specific endonuclease, StuI, produced by Streptomyces tubercidicus KCC S-0054, was identified and partially purified. StuI recognizes the hexanucleotide "palindromic" sequence (Formula: see text), and cleaves it at the middle, producing blunt ends.     

GGC and StuI polymorphism on the androgen receptor gene in endometrial cancer patients

Androgens have an anti-proliferative effect on endometrial cells. Human androgen receptor (AR) gene contains two polymorphic short tandem repeats of GGC and CAG, and a single-nucleotide polymorphism on exon 1 that is recognized by the restriction enzyme, StuI. Prior studies have shown that the lengths of the CAG repeat are inversely and linearly related to AR activity and associated with endometrial cancer. However, little is known about the GGC repeat and the StuI polymorphism of the AR gene. Thus, we investigated whether these AR polymorphisms are risk factors for endometrial cancer. To test this hypothesis, the genetic distributions of these polymorphisms were investigated in blood samples from endometrial cancer patients and healthy controls. The allelic and genotyping profiles were analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and direct DNA sequencing, and analyzed statistically. The GGC repeat was significantly longer in endometrial cancer patients as compared to normal healthy controls. In general, an increased risk of endometrial cancer was found with increasing GGC repeat. The relative risk for the 17 GGC repeat was greater than 4, as compared to controls. However, the StuI polymorphism was not significantly different between patients and controls. The findings suggest that increased numbers of GGC repeat on the AR gene may be a risk factor for endometrial cancer.     

Digestion of centromeric DNA from each human metaphase chromosome by the 6 bp-restriction enzyme StuI

Although 6 bp-restriction endonucleases infrequently cut DNA, we describe in this paper the banding pattern induced by one of them, StuI (AGGCCT), on fixed human chromosomes. This enzyme is the first 6 bp-restriction endonuclease demonstrated to be able to digest human heterochromatin. It causes the extraction of two families of repeated DNA, the alpha satellite DNA and the 3.4 kb HaeIII family (DYZ1). On the other hand, digestions with StuI and with another two enzymes, HinfI and RsaI, have established the distribution of sequences within the heterochromatic block of chromosome 3.     

The impact of StuI digestion in situ on FISH to human chromosomes with satellite DNA probes

Human metaphase chromosomes were digested with StuI and subsequently hybridized in situ using chromosome 9 alphoid DNA and classical satellite III DNA as probes. The data obtained suggest that it is not possible to establish a general rule regarding the cytological effects induced by restriction enzymes in particular chromosome regions and that a number of factors, such as DNA sequences, DNA-protein interaction and enzyme structure, play a role in determining such effects.     

Introducing StuI sites improves vectors for the expression of fusion proteins with factor Xa cleavage sites

The Arg-encoding triplet (AGG) in the recognition sequence Ile-Glu-Gly-Arg for factor Xa can be used to generate a StuI restriction site (AGGCCT) which greatly facilitates the construction of DNA fragments encoding fusion proteins. Following proteolytic cleavage with factor Xa, a protein with the desired N terminus can be obtained     

广东少数民族Morquio综合征StuI位点的RFLP分析

研究广东少数民族群体GALNS基因StuI位点的遗传多态性以及该位点等位基因片段传递的规律,为今后的连锁分析打下基础。采用PCR-RFLP方法,对72例无血缘关系的健康广东少数民族个体的144条染色体和3个家系9位成员的18条染色体进行检测,然后用χ2检验进行统计学处理。等位基因片段D1的频率为0.70, D2为0.30,杂合率为29%,D1、D2的传递规律与理论上预计的完全符合。广东少数民族群体中StuI位点具有多态性,其基因频率（D1和D2）与国外高加索群体的有显著差别,与日本群体及中国南方汉族群体的则无显著差别;而杂合率与高加索群体及日本群体的均有显著差异,但与中国南方汉族群体的则无显著差异。 Abstract:To investigate the genetic polymorphism of the StuI site in the GALNS gene from a national minority population in Guangdong and to study the mode of transmission of alleles,PCR-RFLP was used to analyze 144 chromosomes from 72 Guangdong unrelated healthy national minority individuals,and the genotypes of members in three families.To compare the frequencies and heterzygosity between Guangdong national minority people and Caucasians,Japanese and Chinese Han people by using χ2 test.The frequency of allele D1(295bp) was 0.70,allele D2(138 plus 157 bp)0.30,the heterozygosity was 29%.The genotypes of each member of all families detected were completely agreement with the theorical assessment.The site of StuI in the GALNS gene from national minority population in Guangdong has polymorphism.There is significant difference between Guangdong national minority population and Caucasians in Western countries,but no significant difference was found between Guangdong national minority population and Japanese and Chinese Han population.In addition,there is significant difference between Guangdong national minority population and Caucasians and Japanese in the heterzygosity,but no significant difference between Guangdong national minority population and Chinese Han population.The transmission of alleles was completely in agreement with the Mendelian genetic law.     

Polymerase chain reaction detection of two novel human N-acetylgalactosamine-6-sulfate sulfatase gene polymorphisms by single-strand conformation polymorphism analysis or by StyI and StuI cleavages

A novel polymorphism (6376 G/T) in intron 7 (I7) of the human PROC gene has been identified by direct DNA sequencing. Restriction analysis with the use of mutagenic primers indicate that the allele frequencies are 0.17 (allele T) and 0.83 (allele G), with a calculated heterozygosity of 28%.     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly⁺ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly⁺ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na⁺-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na⁺-independent systems L and Ly⁺remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (V_max) rather than substrate concentration for half-maximal velocity (K_m). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.     

3-Bromo-3-deazaneplanocin and 3-bromo-3-deazaaristeromycin: Synthesis and antiviral activity

As an outgrowth of our program to explore 3-deazaadenine carbocyclic nucleosides, 3-bromo-3-deazaneplanocin (5) and 3-bromo-3-deazaaristeromycin (6) have been synthesized from a readily available cyclopentenol and cyclopentanone and either 4-amino- or 4-chloro-1H-imidazo[4,5-c]pyridine (6-amino- or 6-chloro-3-deazaadenine) in 5 steps and 7 steps, respectively. Antiviral analysis found 5 to display significant activity towards a number of (-)-ssRNA and a few dsDNA viruses. Compound 6 was less active than 5 against selected examples of those viruses affected by 5.     

Ether-linked lipids of Balb/c3T3, SV3T3 and concanavalin A-selected SV3T3 revertant cells

Ether-linked lipids were analyzed in Balb/c3T3, SV3T3 and Concanavalin A-selected SV3T3 revertant cells. The three cell lines were found to contain significant quantities of alk-1-enyl- and alkyl-linked phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and small amounts of alkyldiacylglycerols. Compared to 3T3 cells, SV3T3 cells contain a higher amount of alk-1-enyl-linked PC, while in SV3T3 revertant cells the concentrations of the various ether lipids are similar to those of 3T3 cells. The major difference in the composition of ether groups of SV3T3 cells, compared to 3T3 cells, is an increase of 18:0 accompanied by a decrease of 18:1 in the alk-1-enyl-linked PE and PC. Alk-1-enyl-linked PC of SV3T3 revertant cells also shows an increase of 18:0, while the decrease of 18:1 was not statistically significant.