Mapping of mouse gamma crystallin genes on chromosome 1

Restriction fragments analysis of DNA from mouse-hamster somatic-cell hybrid clones revealed that a mouse gamma crystallin cDNA hybridized to genomic sequences located on mouse chromosome 1. Identification of restriction fragment length polymorphisms (RFLPs) in the gamma crystallin sequences of inbred strains of mice permitted the further localization of the gamma crystallin genes (Cryg) to the proximal region of chromosome 1 closely linked to the loci encoding isocitrate dehydrogenase (Idh-1), a low molecular weight (LM) crystallin protein polymorphism (Len-1), and fibronectin (Fn-1). A single recombinant was observed betweenLen-1 and an RFLP in the gamma crystallin gene family, consistent with the hypothesis thatLen-1 is one of the several structural loci encoding gamma crystallin genes.Len-1 is probably located on the centromeric end of theCryg gene family. Linkage ofIdh-1, Cryg, andFn-1 in mice extends the syntenic relationship of those loci to the human, bovine, and rodent genomes and may define a chromosomal region that is generally conserved among mammals. The map position ofCryg, near the eye lens obsolescence (Elo) locus, was confirmed by the discovery that the restriction fragment patterns of gamma crystallin sequences differed between strain C3H/HeJ and the congenic anophthalmic mutant strain, C3H.Elo. Therefore, the gamma crystallin genes were contransferred with the mutantElo gene in the derivation of C3H.Elo. The results establish that LEN-1 is a marker for the gamma crystallin gene family, position the gamma crystallin gene family relative to other markers on mouse chromosome 1, and provide additional evidence that theElo mutation is encoded at a locus closely linked to the gamma crystallin gene cluster. This study found no evidence of recombination hot spots within the gamma crystallin gene cluster.     

Deletion mutants as probes for localizing regions of subunit interaction in cAMP-dependent protein kinase

The regulatory subunit of cAMP-dependent protein kinase has a well-defined domain structure, and recombinant DNA techniques have been used to define further the functional properties that are associated with each domain. Our initial question was to define the minimal structural unit that is required for forming a stable complex with the catalytic subunit that will still bind and hence be dissociated by cAMP. To answer these questions, the entire second cAMP-binding domain was deleted using oligonucleotide-directed mutagenesis to introduce a premature stop codon at Trp260. This mutation results in the expression of a stable protein with an Mr of 38,000 based on polyacrylamide gel electrophoresis. The resulting mutant protein is a dimer; and like the native R-subunit, the two protomers of the dimer are cross-linked by disulfide bonds at the amino terminus. The mutant R-subunit binds 1 mol of cAMP/monomer based on equilibrium dialysis. The Kd(cAMP) was 25 nM, which is slightly higher than the Kd(cAMP) for the native R-subunit. The removal of the second cAMP domain does not prevent aggregation with the catalytic subunit, and the inactive holoenzyme complex that is formed in the absence of cAMP can still be dissociated and consequently activated by cAMP. In conjunction with previous results based on limited proteolysis, it is concluded that the region extending from Arg94 to Lys259 constitutes a structural unit that will be sufficient to interact with the catalytic subunit in a cAMP-dependent manner.     

Generation of hydroxyeicosatetraenoic acids by human inflammatory cells: analysis by thermospray liquid chromatography-mass spectrometry

Arachidonic acid was converted to a series of hydroxyeicosatetraenoic acids (HETEs) by mixed human inflammatory cells following stimulation with the calcium ionophore A23187. HETEs were purified by a simple one-step extraction procedure followed by HPLC. The HPLC was coupled to a Finnigan quadrupole mass spectrometer using the now commercially available thermospray liquid chromatography-mass spectrometry interface. The HPLC eluant was monitored 'on line' by the mass spectrometer. Soft ionisation occurs, generating intense molecular ion species in the negative ion mode (M - H-:m/z 319) for each of the isomeric HETEs. The (M + H+ - H2O) ion at m/z 303 is the major species in the positive ion spectra of HETEs. Mass spectra were obtained on-line post-HPLC for HETEs formed by the human cells, and the HPLC-MS profile compared with that obtained from standards; species corresponding to the 11-, 9- and 5-HETEs were observed.     

Nonlever action of the mandible

This study considers the current concept of the mandible as a lever of the third order. The concept requires a fulcrum, and this function has been ascribed to the condyle region, but it tends to be overlooked that the fulcrum of a third-order lever in this case would sometimes have to bear a considerable stress. Certain changes, attributed to stress, have been observed in anatomical components of the articulation, but they cannot be explained in terms of the lever concept. They are accounted for by the changing anatomical relations in the working and contralateral sides during mandibular function. They arise from minor stress, especially when dental conditions indicate a period of abnormal function.     

Chromosomal localization of Emv-16 and Emv-17, two closely linked ecotropic proviruses of RF/J mice.

Emv-16 and Emv-17, the two closely linked ecotropic proviral loci of RF/J mice, have been mapped to chromosome 1 between leaden, ln, and the mouse engrailed homeo-box locus, En-1, by using recombinant inbred strains and conventional backcross analysis.     

Leaf growth of Betula and Acer in simulated shadelight

Summary Leaves of birch (Betula pendula Roth) and sycamore (Acer pseudoplatanus L.) were initiated and grown either in a simulated shadelight (80 mol m^-2 s^-1, R/FR ratio 0.28)/dark photoenvironment or a white light (250 mol m^-2 s^-1, R/FR>1)/dark photoenvironment. Until the leaves were more than 50% expanded, growth rates (measured every 24 h) were the same for both species in both environments. After this time, growth rate slowed and this correlated well with a decrease in wall extensibility (WEX). Birch leaves in shadelight showed reduced surface acidification and were the first to show reduced growth. WEX under these conditions was particularly low.Daily patterns of leaf growth of the two species were very different. Sycamore leaves showed a slightly higher growth rate in the dark than in shadelight, while birch leaves grew more rapidly in shadelight than in the dark. Limitation of growth of sycamore leaves in light may be explained by a very high yield threshold turgor for growth (Y). The daily pattern of leaf growth shown by birch is more difficult to explain but the importance of a possible limitation of growth by solute availability and a diurnal variation in Y are discussed.     

Evolution of the apolipoproteins. Structure of the rat apo-A-IV gene and its relationship to the human genes for apo-A-I, C-III, and E

We have determined the nucleotide sequence of the rat apolipoprotein (apo-) A-IV gene and analyzed its structural and evolutionary relationships to the human apolipoprotein A-I, E, and C-III genes. The rat A-IV gene is 2.4 kilobases in size and consists of three exons (142, 126, and 1157 base pairs) interrupted by two introns (277 and 673 base pairs). The 5'-nontranslated region and most of the signal peptide are encoded by the first exon. Thus, the apo-A-IV gene lacks an intron in the 5'-nontranslated region of its mRNA in contrast to all other known apolipoprotein genes. Sequences coding for amphipathic docosapeptides span both the second and third exons of the rat A-IV gene. We demonstrate that this is also true for the human apolipoprotein genes. This gene family seems to have evolved by the duplication of an ancestral minigene that resulted in the formation of two exons. Thereafter, evolution of these sequences was dominated by intraexonic amplification of repeating units coding for amphipathic peptides. Sequence divergence of these repeats resulted in the functional differentiation of the apolipoproteins. However, conservation of the fundamental amphipathic pattern allowed members of this protein family to retain their lipid-binding properties.     

Probing the structure of cytoplasm

We have used size-fractionated, fluorescent dextrans to probe the structure of the cytoplasmic ground substance of living Swiss 3T3 cells by fluorescence recovery after photobleaching and video image processing. The data indicate that the cytoplasm of living cells has a fluid phase viscosity four times greater than water and contains structural barriers that restrict free diffusion of dissolved macromolecules in a size-dependent manner. Assuming these structural barriers comprise a filamentous meshwork, the combined fluorescence recovery after photobleaching and imaging data suggest that the average pore size of the meshwork is in the range of 300 to 400 A, but may be as small as 200 A in some cytoplasmic domains.     

Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

An expression vector has been constructed for the type I regulatory subunit of cAMP-dependent protein kinase. A cDNA clone for the bovine RI-subunit has been inserted into pUC7. When Escherichia coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-azidoadenosine 3':5'-mono[32P]phosphate following transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of isopropyl-beta-D-thiogalactopyranoside. R-subunit accumulated in large amounts only in the stationary phase of growth, and the addition of isopropyl-beta-D-thiogalactopyranoside during the log phase of growth actually blocked the accumulation of R-subunit. Maximum expression (20 mg/liter) was achieved when E. coli 222 was transformed with the RI-containing plasmid. E. coli 222 is a strain that contains two mutations; it is cya- and also has a mutation in the catabolite gene activator protein (crp) that enables the protein to bind to DNA in the absence of cAMP. The expressed RI-subunit was a soluble, dimeric protein, and no significant proteolysis was apparent in the cell extract. The purified RI-subunit bound 2 mol of cAMP/mol of R monomer, reassociated with C-subunit to form holoenzyme, and migrated as a dimer on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis, yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties, the expressed protein was indistinguishable from RI purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z' gene of the vector. This NH2-terminal sequence was confirmed by amino acid sequencing.