Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein ILF.

Recently we isolated a cellular DNA binding protein, designated interleukin enhancer binding factor (ILF), that binds to purine-rich regulatory motifs in both the HIV-1 LTR and the IL2 promoter. Further analysis of the ILF gene reveals the existence of two mRNA species, both of which encode proteins containing the recently described fork head DNA binding domain. Gel retardation analysis demonstrates that the portion of the ILF protein with homology to the fork head domain is sufficient to mediate DNA binding to a number of related purine-rich sequences. ILF mRNA is expressed constitutively in both lymphoid and nonlymphoid tissues. Chromosomal mapping localizes the ILF gene to human chromosome 17q25, which is a site of chromosomal translocations in some cases of human acute myelogous leukemias. These studies further characterize the structure of the cellular DNA binding protein ILF and may prove valuable in the molecular analysis of possible translocations affecting this gene.     

Metabolism of [4-14C]estrone in hamster and rat hepatic and renal microsomes: species-, sex- and age-specific differences.

The metabolism of [4-14C]estrone (E1) was examined in liver and kidney microsomes of adult castrated male and ovariectomized female hamsters and rats and in neonatal and immature hamster renal microsomes. In castrated male hamster liver microsomes, E1 was metabolized extensively to six major metabolites; 15 beta-hydroxyestrone, 7 alpha-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone, 2-hydroxyestrone, and delta(9,11)-dehydroestrone, and a nonpolar fraction. Six minor metabolites of E1 were also detected. In contrast, kidney microsomes derived from castrated male hamsters metabolized E1 to mainly 17 beta-estradiol, 2- and 4-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone and one monohydroxyestradiol metabolite. However, 16 alpha-hydroxyestrone was not detected. A variable, but low amount of estriol was also found. Interestingly, the quantity of 2-hydroxyestrone found in kidney microsomes of the hamster represented 26% of the total amount of metabolites formed, whereas in liver microsomes, only 9% of the overall metabolism resulted in the formation of 2-hydroxyestrone. The ability of kidney microsomes of female ovariectomized hamsters and two different rat strains to metabolize E1 was 5.9- and 9.4-fold lower, respectively, compared to renal microsomes of male castrated hamsters. The onset of oxidative metabolism in newborn hamster kidneys during development was also assessed. The results indicate that the oxidative metabolism of [14C]E1 in renal microsomes of newborn hamsters was 20-fold less than in kidney microsomes of adult hamsters. While catechol E1 metabolites were essentially negligible in hamster kidneys of these ages, it was evident that the conversion of E1 to estradiol via 17 beta-hydroxysteroid dehydrogenase resembles levels seen in the adult animals. Between the age of one and two months, the male hamster kidney exhibited the capacity to metabolize E1 at levels seen in fully mature adult hamsters.     

Urea-induced transformation of native estrogen receptor and evidence for separate DNA-and ATP-binding sites

We have investigated the involvement of hydrophobic receptor domains during transformation of the native estrogen receptor to a form(s) with high affinity for immobilized DNA and ATP. In the presence of 6 M urea the intact estrogen-receptor complex was completely (greater than 90%, n = 12) transformed into a DNA-binding configuration but only partially (35-45%, n = 8) transformed into an ATP-binding state. Similar experiments performed with unliganded receptor preparations further distinguished the receptor's DNA and ATP binding properties. While the urea-induced increase in receptor affinity for DNA-agarose was estrogen-dependent, the urea-induced increase in affinity for ATP-agarose was steroid-independent. This is the first direct evidence that hydrophobic receptor domains may be involved in the steroid-dependent exposure of the DNA binding site. This event is partially reversible and suggests that electrostatic interactions alone may not be sufficient to accurately describe receptor recognition of specific DNA acceptor sites.     

Duck hepatitis B virus can tolerate insertion, deletion, and partial frameshift mutation in the distal pre-S region.

In-frame and frameshift mutations were introduced into the pre-S region (1,212 base pairs) of duck hepatitis B virus. The in-frame mutants retained the inserted 12 nucleotides, while the frameshift mutants either reverted to wild type or exhibited a 10-nucleotide compensatory deletion downstream of the original mutation site. Thus, although duck hepatitis B virus has a compact and highly economical genome organization, it can replicate despite alterations of up to 9 amino acid codons in the pre-S and P open reading frames.     

Cloning of two adult hamster globin cDNAs (alpha and beta major).

A cDNA library was prepared from poly(A) mRNA extracted from adult anemic hamster spleen erythroid cells. cDNA clones containing inserts coding for adult alpha and beta major globin chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNA and (b) nucleotide sequence analysis of the inserts and comparison to the adult globin cDNAs of mouse, rabbit and human. Availability of sequences for embryonic (Li et al. (1992) Biochim. Biophys. Acta 1130, 218-220) and adult globin cDNAs (this report) will aid in investigations of the molecular mechanisms involved in the globin ontogeny of hamsters.     

Ⅱ型限制性核酸内切酶NcrⅠ的研究

 在肉色诺卡氏菌C-212株Nocardia carnea C-212中筛选到一种Ⅱ型限制性核酸内切酶NcrⅠ,经与BglⅡ的λDNA降解物的酶谱比较,以及酶识别特异性和切割位点的检测,证明了NcrⅠ是已知的限制酶BglⅡ的同切限制酶,而且其切割位点也与BglⅡ相同,其为: