Nuclear matrices containing residual DNA were isolated from chicken erythrocytes after extraction of purified nuclei with buffered 2 M NaCI. After further purification of this residual DNA, it was found to contain high concentrations of -Wglobin gene sequences as assayed by dot hybridization with 32P-labeled nick-translated pHB1001. Electron microscopy of a random sample of this residual DNA fraction shows the DNA to be intimately associated with protein at various intervals. A hypothesis for enrichment of active genes in residual DNA from purified chromatin or in nuclear matrix DNA is also discussed.During the past decade many diverse experimental strategies have been applied to the study of eukaryotic transcriptional regulation by the nonhistone chromosomal proteins (1). In spite of all of these efforts, the nonhistones presumably responsible for tissue-specific transcription remain elusive. Studies on DNA sequences have provided evidence suggesting that the nucleotide sequences offlanking and intervening sequences are essential for correct gene expression (2). Nevertheless, analysis of restriction enzyme digests of chicken DNA for ,-globin (3), ovalbumin (4), conalbumin (5), and lysozyme (6) have shown that these genes appear to be indistinguishable between tissues where the genes are either active or inactive. Furthermore, comparison of the sequences around the putative promoter sites of the ovalbumin gene (7) cloned from the DNA of producer and nonproducer tissues revealed no differences. Thus it is reasonable to suggest that the nucleotide sequence alone is insufficient for genomic activation of tissue-specific transcription. Although gene-specific nonhistone proteins have not yet been found in chromatin obtained from specialized tissues, results supporting that possibility have been reported (8)(9)(10)(11)(12)(13).Of the total nuclear proteins, the insoluble nonhistones are significant in their content of tissue-specific proteins (14), in the structure of the nuclear matrix (15), and in their association with transcriptionally active genes (16). Recently, studies by Pettijohn's group also suggested that an insoluble nonhistone protein of high molecular weight appears to be specific to cells undergoing active proliferation (17). Previous results from our laboratory have shown that it is possible to isolate from purified chromatin a DNA fraction significantly enriched in tissue-specific gene sequences (18)(19)(20). We suspect that this DNA, which we have designated DNA-P, is similar to that DNA found associated with the nuclear matrix. Transcribed sequences are associated with the nuclear matrix (21), and these sequences may form the base of the DNA loops in which they reside (22). We show by dot hybridization that the insoluble DNA fraction isolated from chicken erythrocyte nuclear matrices contains high concentrations of f3-globin gene sequences, although this gene is known to be inactive in mature chicken erythrocytes (23). MATERIALS AND METHODSIsolation of DNA Associated with Nonhistones from th...
Extraction of chicken reticulocyte and erythrocyte chromatins with 2 M NaCl yields a small fraction (about 5%) of the total DNA which is very tightly bound to a class of nonhistone chromatin proteins (DNA-P). This DNA fraction has previously been shown to be significantly enriched in active gene sequences. The proteins associated with reticulocyte and erythrocyte DNA-P were analyzed by two-dimensional gel electrophoresis. Reticulocyte DNA-P yield predominantly three major proteins, designated G1, G2, and G3 with relative masses of 80 000, 50 000, and 58 000, respectively. Erythrocyte DNA-P show only two proteins which appear to be similar to the reticulocyte G1 and G2 proteins, except in much reduced quantities as revealed by two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis of the three reticulocyte proteins revealed that the ratio of acidic to basic amino acid residues increased in the order G1 less than G2 less than G3, while the respective isoelectric points also increased in that order.
It is shown that the maximum average-data-collection-speed (ADCS) of multisection 2D hybrid-RARE sequences is independent of TR and TEeff, and a monotonically increasing function of echo-train-length (ETL). This result was used in the design of an optimized T1-weighted hybrid-RARE sequence that produces 20 images of the abdomen in 31 s divided into four breath-hold periods. The resulting ADCS is 58 lines in k-space per second. Twenty-four subjects (2 healthy volunteers and 22 patients) were imaged with a protocol that also included: (a) breath-hold T1-weighted FLASH which acquires data at 34 lines in k-space per second (49 s scan time), and (b) T1-weighted conventional spin-echo (9:44 minutes scan time) with respiratory compensation. The experiments show that this T1-weighted-hybrid-RARE sequence has: (1) a level of T1 weighting that is comparable with the conventional sequences, (2) very low vulnerability to susceptibility artifacts, (3) high data acquisition efficiency, and (4) higher SNR than T1-weighted-FLASH. In conclusion, the T1-weighted-hybrid-RARE sequence described herein is an efficacious and reproducible technique for rapid imaging of the upper abdomen during suspended respiration.
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