A comprehensive examination of protein sequences for evidence of internal gene duplication

Summary We have implemented a routine procedure for screening protein sequences for evidence of intragenic duplications. We tested 163 protein sequences representing 116 superfamilies of unrelated proteins. Twenty superfamilies contain proteins with internal gene duplications. The intragenic duplications detected can be divided into two major types. (1) One or more duplications of all or part of a gene produce a protein with two or several detectable regions of sequence homology. Sequences from 18 superfamilies contained this type of duplication. (2) Repeated reduplication of a small DNA segment can produce a protein that is repetitive over most of its length. Three superfamilies contain such repetitive sequences. We also investigated the limits of detection of ancient duplications using sequences derived by random mutation of a model sequence consisting of ten 10-residue repeats. The original repetitive nature of the sequence was usually detected after 250 point mutations even though the ancestral segment could not be accurately reconstructed.     

Characterization of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii.

The kinetic properties of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) partially purified from the soil amoeba Acanthamoeba castellanii have been studied. The transferase phosphorylated the lysosomal enzymes uteroferrin and cathepsin D 3-90-fold better than nonlysosomal glycoproteins and 16-83-fold better than a Man9GlcNAc oligosaccharide. Deglycosylated uteroferrin was a potent competitive inhibitor of the phosphorylation of intact uteroferrin (Ki of 48 microM) but did not inhibit the phosphorylation of RNase B or the simple sugar alpha-methylmannoside. Deglycosylated RNase (RNase A) did not inhibit the phosphorylation of RNase B or uteroferrin. These results indicate that purified amoeba GlcNAc-phosphotransferase recognizes a protein domain present on lysosomal enzymes but absent in most nonlysosomal glycoproteins. The transferase also exhibited a marked preference for oligosaccharides containing mannose alpha 1,2-mannose sequences, but this cannot account for the high affinity binding to lysosomal enzymes. A. castellanii extracts do not contain detectable levels of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, the second enzyme in the biosynthetic pathway for the mannose 6-phosphate recognition marker. We conclude that A. castellanii does not utilize the phosphomannosyl sorting pathway despite expression of very high levels of GlcNAc-phosphotransferase.     

Origins of immunoglobulin heavy chain domains

Summary Using computer programs that analyze the evolutionary history and probability of relationship of protein sequences, we have investigated the gene duplication events that led to the present configuration of immunoglobulin C regions, with particular attention to the origins of the homology regions (domains) of the heavy chains. We conclude that all of the sequenced heavy chains share a common ancestor consisting of four domains and that the two shorter heavy chains, alpha and gamma, have independently lost most of the second domain. These conclusions allow us to align corresponding regions of these sequences for the purpose of deriving evolutionary trees. Three independent internal gene duplications are postulated to explain the observed pattern of relationships among the four domains: first a duplication of the ancestral single domain C region, followed by independent duplications of the resulting first and last domains. In these studies there was no evidence of crossing-over and recombination between ancestral chains of different classes; however, certain types of recombinations would not be detectable from the available sequence data.     

The isolated ER-Golgi intermediate compartment exhibits properties that are different from ER and cis-Golgi

A procedure has been established in Vero cells for the isolation of an intermediate compartment involved in protein transport from the ER to the Golgi apparatus. The two-step subcellular fractionation procedure consists of Percoll followed by Metrizamide gradient centrifugation. Using the previously characterized p53 as a marker protein, the average enrichment factor of the intermediate compartment was 41. The purified fraction displayed a unique polypeptide pattern. It was largely separated from the rough ER proteins ribophorin I, ribophorin II, BIP, and protein disulfide isomerase, as well as from the putative cis-Golgi marker N-acetylglucosamine-1-phosphodiester-alpha-N-acetylglucosaminidase, the second of the two enzymes generating the lysosomal targeting signal mannose-6-phosphate. The first enzyme, N-acetylglucosaminylphosphotransferase, for which previous biochemical evidence had suggested both a pre- and a cis-Golgi localization in other cell types, cofractionated with the cis-Golgi rather than the intermediate compartment in Vero cells. The results suggest that the intermediate compartment defined by p53 has unique properties and does not exhibit typical features of rough ER and cis-Golgi.     

Localization of the human type 5, tartrate-resistant acid phosphatase gene by in situ hybridization

We report the localization of the gene for the human type 5, tartrate-resistant, iron-containing acid phosphatase isoenzyme (HGM designation ACP5) to chromosome 15 (15q22-q26) using the technique of in situ hybridization to metaphase chromosomes. We have localized this gene using peripheral blood chromosomes obtained from both a normal male and an individual carrying an unbalanced translocation involving chromosome 15 [45,XY,-15,-18,+der(18)-t(15;18)(q13;p11)]. In addition, we have demonstrated the utility of employing a standard fluorescent staining technique (distamycin/DAPI) to an emulsion-coated, Wright-stained, and destained chromosome preparation.     

Uteroferrin contains complex and high mannose-type oligosaccharides when synthesized in vitro

Mature uteroferrin (Uf; M = 35,500) is a progesterone-induced acid phosphatase secreted by the pig uterus. It contains a single, unphosphorylated, high mannose-type oligosaccharide. Endometrial explants cultured in vitro secrete Uf with a M of 37,000 (37k Uf) having phosphorylated high mannose oligosaccharides. In this report we demonstrate that 37k Uf contains two N-linked oligosaccharides which are a mixture of complex and high mannose-type oligosaccharides. The complex-type glycopeptides are biantennary and a portion may be fucosylated on the GlcNac of the chitobiose core proximal to the peptide. Only a portion of the high mannose-type oligosaccharides are phosphorylated. The remainder appear to be typical Man_6-4GlcNac₂ oligosaccharides found on mature Uf.Abbreviations Uf Uteroferrin - ConA Concanavalin A - WGA Wheat Germ Agglutinin - endoH endo--N-acetylglucosaminidase H - SDS Sodium Dodecyl Sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS     

Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications

A class of N-substituted quinoline compounds has been introduced recently for the fluorescence measurement of Cl concentration in biological preparations. The most Cl-sensitive compound was 6-methoxy-N-[3-sulfopropyl] quinolinium with peak excitation and emission wavelengths of 350 and 442 nm and a Stern-Volmer constant for quenching by Cl of 118 M-1. Six water-soluble quinoline derivatives were synthesized and characterized for the purposes of increasing Cl sensitivity, adding ester functions for cell trapping, and red-shifting the fluorescence peak wavelengths. Acetic acid ester functions were added at the N-, 2-, and 6-positions of the quinoline ring. The best ester compound, N-(6-methoxyquinolyl)acetoethyl ester (MQAE), was water soluble (270 g/liter at 23 degrees C; octanol:H2O partition coefficient of 0.009), had a high Cl sensitivity (Stern-Volmer constant 200 M-1), peak excitation and emission wavelengths of 355 and 460 nm, a fluorescence lifetime of 21.6 ns, and a molar absorbance of 4850 M-1 cm-1 (320 nm). MQAE fluorescence was not altered by the physiological anions HCO3, SO4, and PO4, by cations, or by pH. MQAE was used to measure chloride transport in liposome membranes and in cultured LLC-PK1 cells in monolayer; MQAE leaked out of cells less than 20% in 60 min at 37 degrees C. The physical, optical, and anion quenching properties for the series of ester compounds were determined to establish a set of structure-activity correlates.     

Patterns in ontogeny of human trabecular bone from SunWatch Village in the Prehistoric Ohio Valley: general features of microarchitectural change

Although adult skeletal morphological variation is best understood within the framework of age-related processes, relatively little research has been directed towards the structure of and variation in trabecular bone during ontogeny. We report here new quantitative and structural data on trabecular bone microarchitecture in the proximal tibia during growth and development, as demonstrated in a subadult archaeological skeletal sample from the Late Prehistoric Ohio Valley. These data characterize the temporal sequence and variation in trabecular bone structure and structural parameters during ontogeny as related to the acquisition of normal functional activities and changing body mass. The skeletal sample from the Fort Ancient Period site of SunWatch Village is composed of 33 subadult and three young adult proximal tibiae. Nondestructive microCT scanning of the proximal metaphyseal and epiphyseal tibia captures the microarchitectural trabecular structure, allowing quantitative structural analyses measuring bone volume fraction, degree of anisotropy, trabecular thickness, and trabecular number. The microCT resolution effects on structural parameters were analyzed. Bone volume fraction and degree of anisotropy are highest at birth, decreasing to low values at 1 year of age, and then gradually increasing to the adult range around 6-8 years of age. Trabecular number is highest at birth and lowest at skeletal maturity; trabecular thickness is lowest at birth and highest at skeletal maturity. The results of this study highlight the dynamic sequential relationships between growth/development, general functional activities, and trabecular distribution and architecture, providing a reference for comparative studies.