Arabidopsis PAI gene arrangements, cytosine methylation and expression.

Previous analysis of the PAI tryptophan biosynthetic gene family in Arabidopsis thaliana revealed that the Wassilewskija (WS) ecotype has four PAI genes at three unlinked sites: a tail-to-tail inverted repeat at one locus (PAI1-PAI4) plus singlet genes at two other loci (PAI2 and PAI3). The four WS PAI genes are densely cytosine methylated over their regions of DNA identity. In contrast, the Columbia (Col) ecotype has three singlet PAI genes at the analogous loci (PAI1, PAI2, and PAI3) and no cytosine methylation. To understand the mechanism of PAI gene duplication at the polymorphic PAI1 locus, and to investigate the relationship between PAI gene arrangement and PAI gene methylation, we analyzed 39 additional ecotypes of Arabidopsis. Six ecotypes had PAI arrangements similar to WS, with an inverted repeat and dense PAI methylation. All other ecotypes had PAI arrangements similar to Col, with no PAI methylation. The novel PAI-methylated ecotypes provide insights into the mechanisms underlying PAI gene duplication and methylation, as well as the relationship between methylation and gene expression.     

Cathepsins B and H from porcine spleen. Purification, polypeptide chain arrangements, and carbohydrate content

Procedures for the purification of cathepsins B and H from porcine spleens have been described. The purified porcine cathepsin B (Mr = 27,000) is predominantly a two-chain enzyme with a heavy chain (Mr = 22,000) and a light chain (Mr = 5,000). It also contains two minor forms of cathepsin B with different chain structures. Porcine cathepsin H is a single-chain enzyme with a molecular weight of 25,000. The carbohydrate analyses showed that these enzymes were glycoproteins. A glycopeptide containing 3 amino acids, 2 glucosamines, and 6 mannoses was isolated from cathepsin H. Proton NMR studies revealed that it contained a mixture of 4 high mannose-type of oligosaccharides characteristic of those found on lysosomal enzymes. The carbohydrate of cathepsin B consisted of a single residue of glucosamine and trace mannose. This sugar content is in agreement with the finding that about 80% of the porcine spleen cathepsin B contained a single N-acetylglucosamine while 20% of the enzyme contained a 5-sugar oligosaccharide (Takahashi, T., Schmidt, P. G. and Tang, J. (1984) J. Biol. Chem. 259, 6059-6062). Thus, the studies on carbohydrate contents also indicated the good purity of the enzymes.     

Gene arrangements and phylogeny in the class Proteobacteria.

A simple method is presented for reconstructing phylogenetic trees on the basis of gene transposition. It is shown that differences in gene arrangements among genomes could allow us to determine whether a gene transposition event has occurred before or after species divergence from parsimonious considerations. The method is applied to evolutionary relationships among the bacterial class Proteobacteria, for which complete genomic sequences most densely accumulate and comprehensive gene order comparisons are possible. We were able to infer the emergence order of proteobacterial subclasses as epsilon-->beta-->gamma. This order is consistent with sequence-based inferences, which conversely confirms the usefulness of the approach presented here.     

Molecular and physical arrangements of human DNA in HRAS1-selected, chromosome-mediated transfectants.

We used mitotic chromosomes isolated from a human EJ bladder carcinoma cell line for morphological transformation of mouse C127 cells. These chromosome-mediated transformants were analyzed for cotransfer of markers syntenic with c-Ha-ras-1 on human chromosome 11. We also used cloned, dispersed human DNA repeats, in a general mapping strategy, to quantitate the amounts and molecular state of human DNA transferred along with the activated c-Ha-ras-1 gene. In situ hybridization was used to visualize the physical state of the transfected human chromatin. The combined use of these various techniques revealed the occurrence of both chromosomal and DNA rearrangements. However, our analysis also demonstrated that, in general, very substantial lengths of DNA are transferred intact. Closely linked markers are likely to cosegregate. Therefore, these transformants should be invaluable sources for the complete molecular cloning of isolated fragments of the short arm of human chromosome 11.     

Hierarchical, parallel, and serial arrangements of sensory cortical areas: connection patterns and functional aspects.

Recent studies have led to a better understanding of the organization and connections of somatosensory and visual cortex in a number of mammalian species. Lesion studies have provided information on the significance of particular connections. The variable effectiveness of cortical lesions in deactivating target areas suggests that serial processing may be emphasized in higher primates.     

Comparative studies of two cathepsin B isozymes from porcine spleen. Isolation, polypeptide chain arrangements, and enzyme specificity

Our previous studies on carbohydrate structures of purified porcine spleen cathepsin B indicated that there are two cathepsin B isozymes, each containing a different carbohydrate (Takahashi, T., Schmidt, P.G., and Tang, J. (1984) J. Biol. Chem. 259, 6059-6062). We have now isolated these two enzymes and carried out a comparative study on their structures and enzymic properties. The major isozyme (CB-I) is a two-chain enzyme (Mr = 28,000) with a light chain (Mr = 5,000) and a heavy chain (Mr = 23,000), whereas the minor enzyme (CB-II) is a single chain enzyme (Mr = 27,000). The NH2-terminal amino acid residues of CB-I were leucine and valine for the light and heavy chain, respectively. However, the NH2-terminal residue of CB-II was not available for automated Edman degradation. In addition, peptide mapping experiments indicated a difference in the primary structure of these two proteins. Despite such structural differences, they are similar in many enzymic properties. CB-I was more catalytically efficient than CB-II toward synthetic substrates, except for the substrate benzoyl-L-arginine beta-naphthylamide for which the relative catalytic efficiency is reversed. Both isozymes degraded glucagon by a dipeptidyl carboxypeptidase activity. Under the same conditions, CB-I was 4-5 times more efficient than CB-II. The results indicate that the cathepsin B isozymes are two separate gene products, but they are similar in enzymic properties.     

Common spatial arrangements of backbone fragments in homologous and non-homologous proteins.

We have developed a new method of detecting common spatial arrangements of backbone fragments in proteins. This method allows corresponding fragments to occur in a different order in respective amino acid sequences. We applied this method to detect structural similarities between an acid protease, endothiapepsin, and all other proteins in the protein data bank. Significant similarities were found not only with other acid proteases but also with virus proteases and with proteins having different functions. The possible biological meaning of these similarities is discussed.     

Molecular arrangements of sphingolipids. The monolayer behaviour of ceramides

A series of synthetic ceramides have been studied at the air-water interface by recording the surface pressure-area isotherms at continuous compression.

Ceramides that contain a 4,5-trans-double bond in the long chain base were found to condense into a close-packed arrangement with vertical chains already at a very low surface pressure. The corresponding saturated compounds adopt a similar close packed arrangement only at high surface pressure. At 30 dynes/cm, a lateral pressure representative of biological membranes, the area per molecule and compressibility was further found to depend on the number and configuration of the hydroxyl groups. The presence of a 2-D-hydroxyl group in the fatty acud generally promotes the condensation. A similar effect is observed if the long chain base contains a 4-D-hydroxyl group. Cis-double bonds or methyl branches in the fatty acid chain, which increase the space requirement, limit the lateral interaction of the polar group. However the 15-cis-double bond of nervonic acid can be accommodated without any distortion of the close-packing arrangement.     

Molecular arrangements in sphingolipids. The crystal structure of cerebroside

The single crystal analysis of a complex, membrane glycolipid is described. Cerebroside (β-D-galactosyl-N-(2-D-hydroxyoctadecanoyl)-D-dihydrosphingosine, $\text{C}{}_{42}\text{H}{}_{32}\text{O}{}_9\text{N}\text{·}\text{1}\text{2}\text{C}{}_2\text{H}{}_5\text{OH}$) — an important constituent of plasma membranes — crystallizes in the monoclinic spacegroup P2, with a = 11.202, b = 9.262, c = 46.46 A and β = 99.00^°. There are two independent molecules in the asymmetric unit partly related by a non-crystallographic symmetry. The structure was determined by direct methods and refined to R = 0.116.The molecules pack in a typical bilayer arrangement with adjacent double layers separated by ethanol molecules of crystallization. The planes of the sugar rings are turned almost parallel to the layer interface which gives the molecules the shape of a shovel. Together with the polar ceramide part, the galactose head groups form an extensive lateral network of hydrogen bonds within the polar region of each layer. The chains tilt by an angle of 49^° towards this polar boundary. A parallel stacking of the chains is achieved by a bend of the sphingosine chain as far up as carbon atom 5 and 6 in the two independent molecules. The lateral hydrocarbon chain packing is of an earlier unknown hybrid type (HS2). Chains with parallel zigzag planes are arranged in pleated shoets. These sheets contain alternatively fatty acid and sphingosine chains which have a mutually perpendicular chain plane orientation.     

Molecular arrangements in sphingolipids. Thermotropic phase behaviour of tetracosanoylphytosphingosine

The phase behaviour of a ceramide species containing C₁₈-phytosphingosine and C₂₄-fatty acid was studied by X-ray diffraction methods, in order to elucidate the packing principles of lipids with unequally long hydrocarbon chains. Six solid phases were observed. In five of them, the ceramide molecules have an extended, V-shaped conformation and pack in single layer arrangements with the sphingosine and fatty acid chains forming separate matrices. Differences between these phases are mainly due to thermotropic changes in packing efficiency and thus in tilt of the hydrocarbon chains. The chain packing undergoes transitions from triclinic (T|) to monoclinic (M|) and hexagonal, and between orthorhombic (O⊥) and hexagonal subcell arrangements, respectively. Only one case was observed, in which the molecules pack with their two chains parallelly stacked in a double layer arrangement in which the long fatty acid tails deeply interdigitate between the two opposite layer halves. In a natural membrane containing different lipids, however, long fatty acid tails probably arrange randomly and contribute to the formation of a liquid hydrocarbon zone in the center of the bilayer.     

Multiple arrangements of the human embryonic zeta globin genes.

Rearrangements which are most readily explained by homologous crossover between misaligned segments of DNA in the region of the human embryonic zeta (zeta) globin genes have been identified in individuals of three different racial origins. These recombination events have resulted in a surprisingly high prevalence of chromosomes with single (0.4%) and triplicated (1.3%) zeta genes with apparently no significant effect on the phenotype.     

Cathepsin D isozymes from porcine spleens. Large scale purification and polypeptide chain arrangements.

Six cathepsin D isozymes have been purified from porcine spleen using a large scale purification procedure. Five isozymes, I to V, have an identical molecular weight of 50,000 and are similar in specific activity. Isozymes I to IV contained two polypeptide chains each. The light and heavy chains have Mr = 15,000 and 35,000, respectively. Isozyme V is a single polypeptide. The molecular weight of the sixth isozyme is about 100,000 and it has only 5% of the specific activity of the other isozymes. On Ouchterlony immunodiffusion, an antiserum formed precipitin lines against the urea-denatured isozyme with Mr = 100,000. This immunoreactivity showed immunoidentity with those formed against other isozymes. The NH2-terminal sequence of light chains was identical for the isozymes. This sequence is homologous to the NH2-terminal sequence of other acid proteases, especially near the region of the active center aspartate-32. The NH2-terminal sequence of the single chain, isozyme V, Is apparently the same as the light chain sequence. The NH2-terminal sequence analysis of the heavy chain from isozyme I produced two sets of related sequences, suggesting the prescene of structural microheterogeneity. The carbohydrate analysis of the isozymes, the light chain, and the heavy chain revealed the presence of possibly four attachment sites, with one in the light chain and three in the heavy chain. Each carbohydrate unit contains 2 residues of mannose and 1 residue of glucosamine. The results suggest that the high molecular weight cathepsin D (Mr = 100,000) is the probable precursor of the single chain (Mr = 50,000), which in turn produces the two-chain isozymes. These are likely in vivo processes.     

Anatomy of bovine mammillitis DNA II. Size and arrangements of the deoxynucleotide sequences.

We previously reported that bovine mammillitis virus (BMV) DNA consists of two covalently linked components designated L and S and estimated to be 71.5 x 10(6) and 15.7 x 10(6) in molecular weight, respectively; the components invert relative to each other, giving rise to four equimolar populations differing soley in the relative orientation of the two components. We now report that (i) BMV DNA has a contour length corresponding to a molecular weight of 89 x 10(6). (ii) Component L consists of a unique sequence (Ul) bracketed by sequences ab and its inverted repeat b'a', estimated to be of molecular weights 66.1 x 10(6), 2.7 x 10(6), and 2.7 x 10(6), respectively. (iii) Component S consists of a unique sequence (Us) bracketed be sequence ca and its inverted repeat a'c', estimated to be of molecular weights 8.3 x 10(6), 3.7 x 10(6), and 3.7 x 10(6), respectively. (iv) The a sequences present at the termini of a complete linear molecule (abUlb'a'a'c'Usca) are arranged in tandem so that the DNA can circularize after limited digestion with arranged in tandem so that the DNA can circularize after limited digestion with lambda 5'-exonuclease. The size of the a sequences was estimated to be 0.7 x 10(6) in molecular weight. (v) At least portions of the a sequences are repeated in an inverted orientation immediately adjacent to or near the a sequence. Thus, BMV DNA mimics herpes simplex virus type 1 DNA with respect to the arrangement but not size of deoxynucleotide sequences. The evolutionary relationship of BMV DNA relative to other herpesvirus DNAs is discussed.     

Linnaeus's natural and artificial arrangements of plants

Linnaeus's artificial and natural arrangements of plants are examined using a Spearman rank coefficient (which is explained) on his presentations of his own and others' arrangements in the Classes plantarum and elsewhere. There is little alteration in his successive artificial arrangements. In contrast, between 1751 and 1764 his natural arrangements changed considerably, partly in the sequences of genera within orders but mostly by rearrangement of the orders. Comparison with Cesalpino's and Ray's natural arrangements, using the longest-recognized natural groups as signposts, suggests that Linnaeus in his latest natural arrangement (1764) approximated more closely to Ray's. Examination of Linnaeus's successive treatments of certain groups (palms, Zingiberaceae, Hydrocharis-Stratiotes-Vallisneria) and of Giseke's exposition of Linnaeus's lectures on natural groups (1792) shows that Linnaeus was much influenced by habitus and vegetative characters as well as those of the fructification. He recognized orders consisting of a chain of genera linked successively by overall affinity and without any single diagnostic character. Where possible, he preferred characters of the fructification and his ‘secret’ consulting of the habitus is explained as secondary to such characters. It is suggested that in his latest arrangement he approximated more to a scala naturae, as he probably did in zoology about the same time. Within his artificial arrangements Linnaeus kept to sequences of genera as natural as possible. He realized that some groups in his natural arrangements were still artificial, and his aphorism that all genera and species are natural, classes and orders part natural and part artificial, refers to his and others' practice until the natural system could be completed. It is not a statement of the essential natures of these ranks. Linnaeus's distinction in practice between natural and artificial arrangements was less clear-cut than Sachs believed. Linnaeus's rejection of the ancient tree/herb division was empirical, not a reasoned repudiation of an a priori grouping. The tree/herb division could be upheld in his day as obviously natural, not merely accepted on authority.