Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters

We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity). The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur. In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to. When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values. However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons. When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al. (1984). The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed. When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity. The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome. While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers. This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed. Finally, we show that the sequence information, as defined by Schneider et al. (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polypeptides of the Golgi Apparatus of Neurons from Rat Brain

An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100-110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity-purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200-, 150-, 100-110-, and 64-kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40-kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100-110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.     

γ-Hydroxybutyric Acid in Subcellular Fractions of Rat Brain

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.     

Immunoperoxidase confirmation of parathyroid origin of ultrasound-guided fine needle aspirates of the parathyroid glands

It may be difficult to differentiate between cells of parathyroid and thyroid origin in ultrasound-guided fine needle aspirations of the neck region, even in patients with a clinical history of hyperparathyroidism. A parathyroid hormone antibody was used in an immunohistochemical system to confirm a parathyroid origin in fine needle aspirate smears from nine patients with hyperparathyroidism. Immunoperoxidase positivity for parathyroid hormone confirmed a parathyroid origin in six of nine cases and was strongly suggestive, although equivocal, in the remaining three cases. Technical problems included nonuniform staining and background staining.     

Thermochemistry of aqueous solutions of alkylated nucleic acid bases. VI. Enthalpies of hydration of 2-alkyl-9-methyladenines

Enthalpies of solution in water, delta H0sol, and vant'Hoff enthalpies of sublimation, delta H0subl, were determined experimentally for a number of crystalline 2-alkyl derivatives of 9-methyladenine: m2(2,9)Ade, e2m9Ade, pr2m9Ade and but2m9Ade. Standard enthalpies of hydration, delta H0hydr derived from these data were corrected for the calculated cavity terms, delta H0cav, to yield enthalpies of interaction, delta H0int, of the solutes with their hydration shells. The apparent residual contribution of alkyl groups, R, to the enthalpy of interaction delta delta H0int (R) was found to increase linearly with the number of CH2 groups added upon alkyl substitution, whereas this contribution calculated per unit area of the water-accessible molecular surface, SB, of alkyl residues delta delta H0int (R): delta SB(R) appeared constant over the whole series of the compounds investigated. This indicates that alkyl groups substituted at the C(2) carbon atom of the adenine contribute additively to the van der Waals' part of the enthalpy of interaction and do not affect the electrostatic part of the energy of interaction of the solutes with their hydration shells.     

Changes in binucleation and cellular dimensions of rat left atrial myocytes after induced left ventricular infarction

The left atrium of young rats has previously been demonstrated to respond with DNA synthesis and binucleation 11 days after left ventricular infarction. This investigation was designed to examine the hypertrophic response of the left atrial myocyte of the rat at 20 and 60 days after ventricular infarction. Male Sprague-Dawley rats were subjected to left coronary artery ligation (CAL) or sham operation. Following enzymatic separation, left atrial myocytes were examined at 20 and 60 days postoperation for number of nuclei and cellular dimensions (cell length, width and area, and nuclear area). Results demonstrated that the level of binucleation at 20 days (77.3%) and 60 days (71.3%) was nearly twice that observed in sham-operated animals, which were 33.1% binucleated at 20 days and 43.5% binucleated at 60 days. In both mononucleated and binucleated myocytes, the mean lengths, widths, and cell areas from CAL hearts were significantly greater than those of corresponding sham-operated animals. In all cases, these values were larger in binucleated myocytes than in mononucleated cells. The mean area of CAL cells was approximately twice that of sham-operated myocytes. With regard to mean lengths and widths, although both were greater in the CAL animals, there was a decrease in length and increase in width between 20 and 60 days in the CAL group. Mean nuclear areas were significantly greater in CAL myocytes than in those from the sham-operated group. These increases in nuclear number and cellular dimensions of the atrial myocyte are prominent features of the response to the stress imposed by left ventricular infarction.     

Population genetics of coagulant factor IX: frequencies of two DNA polymorphisms in five ethnic groups.

Two frequently used restriction-enzyme polymorphisms (RFLPs) of coagulant F.IX, TaqI and XmnI, have been examined in five ethnic groups: white Americans, black Americans, East Indians, Chinese, and Malays. There is a distinct "cline" in the frequencies of both polymorphisms, from white Americans to Malays. The rarer type 2 alleles of both polymorphisms, in which middle recognition sites are present--and which in our sample reach their highest frequencies in white Americans--are marginally higher in four groups of Europeans previously reported by others. The frequencies of the rarer alleles are significantly higher in Europeans than in black Americans and East Indians, and these alleles are essentially absent in Chinese and Malays. The frequency of heterozygosity diminishes in the same order, being zero in Malays for both polymorphisms. The polymorphisms are in strong linkage disequilibrium, and in all groups the type 1 allele for TaqI is disproportionately accompanied by the type 1 allele for XmnI. The paucity of type 2 alleles and the low rate of heterozygosity in four non-European groups suggest that the polymorphisms will be of little diagnostic value south of Gibraltar and east of Suez. This prediction is confirmed by the observed haplotype frequencies in the black American and the Oriental groups.     

Short and plump physique of Mexican-American children

Mexican-American children are shorter but relatively heavier than non-Hispanic whites and blacks. The objectives of this paper are to assess the extent to which this "short and plump" physique occurs in data collected in two national surveys (HANES I and II); to determine variations by age, sex, and socioeconomic status; and to investigate the anthropometric characteristics that may account for the overweight. Three groups, defined on the basis of reported ancestry and observed race, are studied: Mexican-Americans (MEXAME), non-Hispanic Whites, (EURAME), and blacks (BLACK). Short stature was clearly associated with the poverty index (PI) in all three groups. MEXAMEs with a PI greater than 1.6 were similar in stature to EURAMEs at the same income level at ages 1-11 years but not at 12-17 years. On the other hand, MEXAMEs were shorter than BLACKs at all ages and income levels. The body mass index (kg/cm2) and poverty were unrelated. With respect to the anthropometric characteristics examined that are related to the body mass index, MEXAMEs and EURAMEs were similar in sitting height as a proportion of total height, arm muscle and fat areas, and triceps skinfold but different in the following ways: MEXAMEs had narrower elbow but broader bitrochanteric breadths and larger chest circumferences and subscapular skinfolds. Greater upper body dimensions and fatfolds seem to best describe the physique of MEXAMEs. However, in multiple regressions, these anthropometric characteristics failed to account fully for the greater relative weight of MEXAMEs as compared to EURAMEs.     

Hepatic lipase: a member of a family of structurally related lipases

Partial amino acid sequence of rat hepatic lipase was obtained by gas-phase microsequence analysis of proteolytic fragments. Sequence comparison to bovine lipoprotein lipase and porcine pancreatic lipase reveals a highly conserved region existing among these three physiologically distinct lipolytic enzymes. In a stretch of 36 amino acid residues previously reported for pancreatic lipase (De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P. and Rovery, M. (1981) Biochim. Biophys. Acta 671, 129-138), nineteen residues are identical for all three enzymes, whereas 27 of 36 are identical in rat hepatic lipase and bovine lipoprotein lipase. The fact that this primary structural conservation extends to three different animal species emphasizes the conclusion that these lipolytic enzymes comprise a protein family originating from a common ancestral gene.     

Genetic identification of rat liver carboxylesterases isolated in different laboratories

Six carboxylesterases previously isolated from rat liver microsomes, characterized in Brussels and in Kiel, were compared with genetically defined liver esterases of various reference strains using polyacrylamide gel electrophoresis and isoelectric focusing. The six liver carboxylesterases were identified as alloenzymic forms of ES-3, ES-4, ES-8/ES-10 and ES-15 according to the genetic nomenclature recommended by van Zutphen (Van Zutphen, L.F.M. (1983) Transplant. Proceed. 15, 1687-1688). The genetic and biochemical characteristics of the four isoenzymes are summarized, and their identity with several other drug-metabolizing esterases/amidases and lipases of rat liver endoplasmic reticulum is discussed.