A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme

A novel approach is described that permits the introduction of unidirectional deletions into a cloned DNA fragment, in a precisely controlled manner. The method is based on the use of a special vector and a class-IIS restriction endonuclease, BspMI, which produces staggered cuts 4 and 8 nucleotides (nt) to the 3' from its recognition site 5'-ACCTGC-3'. The DNA fragment is inserted into the pUC19-based plasmid, which contains a unique BspMI recognition site, and the appropriate number of cleavage-and-deletion cycles is performed, each cycle removing 4 bp. Since the recognition site is not affected by the BspMI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of BspMI cleavage, removal of the 4-nt single-stranded cohesive ends with mung bean nuclease (MB), and blunt-end ligation to recircularize the plasmid. The shortened plasmid is reintroduced into the host, after one or after several such 4-bp deletion cycles. When DNA is inserted into the multiple cloning site in the lacZ alpha gene, the progress of 4-bp removal can be followed by determining the Lac phenotype, since removal of multiples of 3 bp retains the reading frame while other kinds of deletions distort (or restore) the reading frame. Loss of pre-existing restriction sites or creation of new ones also permits monitoring the progress of the deletion process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Requirement of acetyl-coenzyme A carboxylase kinase for coenzyme A

Phosphorylation and inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase in the presence of ATP and Mg2+ requires coenzyme A. Coenzyme A did not enhance the phosphorylation of alternative substrates of the carboxylase kinase such as protamine or histones. Analogs of coenzyme A were also effective in stimulating the inactivation of carboxylase. The KA of CoA for stimulated carboxylase inactivation was 25 microM. The presence of coenzyme A did not alter the Km of the carboxylase kinase for its substrates, ATP and acetyl-CoA carboxylase. Fluorescence binding studies showed that CoA binds to carboxylase but not to the kinase. The KD of CoA binding to carboxylase is 27 microM. These results indicate that coenzyme A, acting on acetyl-CoA carboxylase, may play an important role in the regulation of the covalent modification mechanism for acetyl-CoA carboxylase.     

Phosphorylation pattern of a 25 Kdalton stress protein from rat myoblasts

Phosphorylation of a 25 Kdalton nuclear stress protein from rat muscle was examined by two-dimensional gel electrophoresis and one-dimensional peptide mapping. These studies show that three 25 Kdalton stress proteins found in stressed rat myoblasts are actually the same protein with charge variation brought about by multiple phosphorylations. Furthermore, the predominant charge variant of 25 Kdalton protein found in cells is dependent on the intensity of the stress applied to cells.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Strategy for trapping intermediates in the folding of ribonuclease and for using 1H-nmr to determine their structures

The major unfolded form of ribonuclease A is known to show well-populated structural intermediates transiently during folding at 0°–10°C. We describe here how the exchange reaction between D₂O and peptide NH protons can be used to trap folding intermediates. The protons protected from exchange during folding can be characterized by ¹H-nmr after folding is complete. The feasibility of using ¹H-nmr to resolve a set of protected peptide protons is demonstrated by using a specially prepared sample of ribonuclease S in D₂O in which only the peptide protons of residues 7–14 are in the ¹H-form. All eight of these protected peptide protons are H-bonded. Resonance assignments made on isolated peptides containing these residues have been used to identify the protected protons. Other sets of protected protons trapped in the ¹H-form can also be isolated by differential exchange, using either ribonuclease A or S. Earlier model compound studies have indicated that H-bonded folding intermediates should be unstable in water unless stabilized by additional interactions. Nevertheless, peptides derived from ribonuclease A that contain residues 3–13 do show partial helix formation in water at low temperatures. We discuss the possibility that specific interactions between side chains can stabilize short α-helixes by nucleating the helix, and that specific interactions may also define the helix boundaries at early stages in folding.     

A mitochondrial protein essential for the formation of the cytochrome c1-c complex. Isolation, purification, and properties

A new mitochondrial protein was isolated to pure form. This protein was indispensable for the formation of the cytochrome c1-c complex; hence, it was provisionally named the hinge protein for formation of the cytochrome c1-c complex, or for simplicity, merely called the hinge protein. The simplest method for the preparation of the pure protein involved essentially pH 5.5 treatment of high purity of "two-band" cytochrome c1 prepared from an improved method. The use of two band cytochrome c1 prepared by an improved method was preferred because the improved method apparently yielded less tight bonding between the heme-containing and colorless protein entities than that from the original methods (King, T. E. (1978) Methods Enzymol. 53, 181-191). The c1-c complex comprised 1 molar equivalent each of the hinge protein, "one-band" cytochrome c1 and cytochrome c. It was demonstrated by gel filtration chromatography that in the absence of the hinge protein, there was no complex formation between cytochromes c and one-band c1. In titration of the complex formed between one-band cytochrome c1 and cytochrome c with the hinge protein present by using the increase of the Soret-Cotton effect as a criterion (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36), a sharp break was observed which showed the three species to be present in equivalent amounts. The hinge protein showed low extinction in the 280 nm region and exhibited poor color value and diffuse character of the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after staining with Coomassie brilliant blue. The molecular weight was found to be (i) 9,800 from sedimentation equilibrium, (ii) 11,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) 23,000 with a Stokes radius of 22.4 A from gel filtration chromatography estimated from a standard curve with proteins of known molecular parameters. The disparities in these data from the actual value of 9,175 from calculations based on amino acid sequence, as previously reported (Wakabayashi, S., Takeda, H., Matsubara, H., Kim, C. H., and King, T. E. (1982) J. Biochem. (Tokyo) 91, 2077-2085), have been discussed.     

Ethnic Elders and American Health Care—A Physician's Perspective

The aging process is a fugue composed of innumerable themes; the theme of “ethnicity” is by far one of its more dominant. Due to the increasing incidence of chronic, progressive infirmity and acute, catastrophic illness, the elderly are thrust into direct contact with the health care systems of their society. The experiences of ethnic elders in American health care situations are fraught with conflict and mutual dissatisfaction with the physician-patient relationship. Both providers and consumers of health care services harbor differing culture-bound perceptions of health, illness and the healing process; these cultural beliefs define personal and professional needs and expectations and notions of how those needs are to be met by others. Both physicians and patients can enhance their communication and their compassion for one another by acknowledgment of cultural differences and by increased willingness to interpret motives and behavior within native context.It behooves us in medicine to examine the cultural traditions underlying our own attitudes, beliefs and values about the aged in a universal sense, as well as in a culturally specific sense, that we may gain insight that will be helpful in serving elderly persons more effectively, and in solving some of the problems inherent in the aging process.     

Melting order of successively longer yeast phenylalanine-accepting transfer ribonucleic acid fragments with a common 5' end

Temperature-jump methods were used to study the kinetics of the helix to coil transition in three fragments of yeast tRNAPhe that share a common 5' terminus (the 5' end of the mature tRNA). Correlation of the extrapolated helix dissociation time constants with NMR exchange broadening results allows assignment of the structural basis of the optical melting transition in the fragments. The results confirm nuclear magnetic resonance findings on these fragments: the 5' 1/4 fragment has no helical structure; the 5' 1/2 fragment contains the D stem; and the 5' 3/5 fragment contains the D stem and the anticodon stem. These are the structures expected if sequential folding of the tRNA during biosynthesis were to occur. The D stem is the last helix to melt in the 5' 3/5 fragment. We suggest that structural elements in addition to the four Watson-Crick base pairs of the D-stem helix are responsible for the anomalously high Tm of that hairpin.