Human saliva as a source of biochemical genetic markers. II. Genetic interpretations and possible utilization.

Human saliva enzymes are compared to analogous blood enzymes. The genetic interpretations for variants of several saliva proteins are reviewed. The possible use of human saliva enzymes and proteins in population genetic studies and disease diagnosis is discussed.     

Double-strand DNA conformation polymorphisms as a source of highly polymorphic genetic markers

The molecular basis for several apparent restriction fragment length polymorphisms of the porcine growth hormone gene was examined through DNA sequence analysis. Electrophoretic and sequence analysis suggest polymorphisms result from 1–3 base substitutions that affect double-strand DNA conformation and electrophoretic mobility. Two allelic forms of the porcine growth hormone 5'flank and four allelic forms of the second exon/intron region were identified. A marker system was developed which combined conformation polymorphisms with HaeII and DdeI RFLPs. Using this system, nine haplotypes were observed in samples from three US swine breeds. The data presented suggest that double-strand DNA conformation can be exploited in base substitution detection and development of highly polymorphic genetic marker systems.     

Amplified Fragment Length Polymorphism (AFLP) as a source of genetic markers for red algae

A recent PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP), was successfully applied to the red alga Chondrus crispus Stackh. This is apparently the first account to describe the application of AFLP methodology to an alga. Six isolates of C. crispus were analyzed by AFLP. A total of twenty-five primer pairs were screened and six primer pairs were selected for further investigation. Both conservative and variable markers were identified within and between populations; some markers were unique to individuals. As such, AFLP should prove useful as a source of genetic markers in algae for applications as diverse as genome mapping to population genetic investigations. This revised version was published online in June 2006 with corrections to the Cover Date.     

Unexpected role for Helicobacter pylori DNA polymerase I as a source of genetic variability

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5'- 3' exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity.     

Blood biochemical polymorphisms as markers for genetic characteristics of wild Spanish and domestic rabbits

Seventeen blood proteins were studied in a sample of 412 Spanish wild rabbits and in 598 domestic rabbits belonging to various breeds. The wild rabbit populations showed a high level of genetic polymorphism. Six loci were monomorphic, while the remaining ten loci were segregating for at least two alleles. Two of the loci that were polymorphic in the wild rabbits were monomorphic in the domestic ones. Wright's inbreeding coefficient in the total Spanish wild rabbit population was F=5.66, indicating subdivision of the total population. Inbreeding coefficients, estimated by Kidd et al.'s method (Anim. Blood Grps, Biochem. Genet. 11: 21–38), differed significantly from zero, being 15.62%, in wild rabbits and 6–12% in domestic breeds, indicating consanguinity.Genetic distances between wild rabbit populations showed that factors other than geographic distance (e.g., bottlenecks, barriers such as rivers, mountains, etc.) may explain the result that a northern population forms a cluster with two central populations whereas the northeastern populations form a different cluster with another central population. Populations of the first cluster are more closely related to the captive populations than others.There are three population clusters of domestic rabbits, namely (1) New Zealand White and a hybrid combination; (2) Spanish Common, Butterfly, Burgundy, and Californian; and (3) Spanish Giant.     

Isolated chromosomes as a new and efficient source of DArT markers for the saturation of genetic maps

We describe how the diversity arrays technology (DArT) can be coupled with chromosome sorting to increase the density of genetic maps in specific genome regions. Chromosome 3B and the short arm of chromosome 1B (1BS) of wheat were isolated by flow cytometric sorting and used to develop chromosome- and chromosome arm-enriched genotyping arrays containing 2,688 3B clones and 384 1BS clones. Linkage analysis showed that 553 of the 711 polymorphic 3B-derived markers (78%) mapped to chromosome 3B, and 59 of the 68 polymorphic 1BS-derived markers (87%) mapped to chromosome 1BS, confirming the efficiency of the chromosome-sorting approach. To demonstrate the potential for saturation of genetic maps, we constructed a consensus map of chromosome 3B using 19 mapping populations, including some that were genotyped with the 3B-enriched array. The 3B-derived DArT markers doubled the number of genetic loci covered. The resulting consensus map, probably the densest genetic map of 3B available to this date, contains 939 markers (779 DArTs and 160 other markers) that segregate on 304 genetically distinct loci. Importantly, only 2,688 3B-derived clones (probes) had to be screened to obtain almost twice as many polymorphic 3B markers (510) as identified by screening approximately 70,000 whole genome-derived clones (269). Since an enriched DArT array can be developed from less than 5 ng of chromosomal DNA, a quantity which can be obtained within 1 h of sorting, this approach can be readily applied to any crop for which chromosome sorting is available.     

Human lung tissue as a source of colony stimulating activity.

Medium conditioned by human lung tissue was found to contain colony stimulating activity (CSA). This material was tested against mouse and human bone marrow as target system. Colony forming units (CFUc) from both species responded and gave rise to clonal growth in agar cultures. This colony formation was dose dependent and the relationship was a sigmoid one. Experiments to determine the molecular weight of human lung derived colony stimulating Factors brought evidence for four active molecular weight fractions with approximately 79000, 40000, 23000 and 2000 daltons. The 23000 dalton fraction activated human cells only, whereas the other fractions were active on both human and mouse bone marrow cells.     

Human stools as a source of viable colonic epithelial cells.

Human stools consist of a mixture of undigested food residues, colonic microflora, and cellular components shed from the walls of the gastrointestinal tract. The cellular components are made up mostly of terminally differentiated colonic epithelial cells. Using a combination of Percoll density gradient centrifugation and countercurrent centrifugal elutriation, it is now possible to recover these cells as an enriched fraction from fresh human stools. Cells can be visualized on heat-fixed smears of the enriched fractions stained with modified Wright's stain. The enrichment process is optimized by following the segregation of eukaryotic cells as determined by an ELISA technique using monoclonal antibodies against human double-stranded DNA. This work, demonstrating the feasibility of isolating intact colonic cells from stools, has important applications as a noninvasive approach to the biology of exfoliated cells from the gastrointestinal tract.     

Hypervariability of simple sequences as a general source for polymorphic DNA markers.

Short simple sequence stretches occur as highly repetitive elements in all eukaryotic genomes and partially also in prokaryotes and eubacteria. They are thought to arise by slippage like events working on randomly occurring internally repetitive sequence stretches. This predicts that they should be generally hypervariable in length. I have used the polymerase chain reaction (PCR) process to show that several randomly chosen simple sequence loci with different nucleotide composition and from different species show extensive length polymorphisms. These simple sequence length polymorphisms (SSLP) may be usefully exploited for identity testing, population studies, linkage analysis and genome mapping.     

Some genetic and biochemical characteristics of a new source of maize opaque-2 mutant

Summary The opaque kernels separated from the F₁ of crosses of our opaque-2 strains with normal inbred lines contained 45, 50 and 74 percent more lysine in the whole kernel than the translucent kernels from the same ear. The opaque kernels from these crosses contained almost the same level of lysine as the parental opaque-2 strains.Some of our opaque-2 strains contain 63 to 122 percent more lysine than the tested dent or flint normal inbred lines. In comparison with opaque-2 Purdue, four opaque-2 strains had almost the same lysine content and a strain labelled as SP-1 No. 15 contained 25 percent more lysine and 73 percent more tryptophan. Some of our opaque-2 strains contained 55 to 100 percent more tryptophan in the whole kernel than opaque-2 Purdue.In contrast to lysine content, the transmission of the tryptophan content from opaque-2 strains to opaque kernels from their crosses shows variability.We found weak positive correlation (r = +0,3057) between lysine and tryptophan content in opaque-2 kernels.Our opaque-2 strains had a higher lysine content in the endosperm than did opaque-2 Purdue 22.1 – 36.6% more, and compared with normal lines they had 109.5 – 142.6% more.It is apparent that a new source of opaque-2 mutant gene, which has the same genetic and biochemical characteristics as the opaque-2 mutant discovered by Mertz, Bates and Nelson (1964), has been found in a completely new, genetically divergent, strain derived from a large number of populations.

---

Zusammenfassung Die vorgenommenen Prüfungen haben gezeigt, daß undurchsichtige Körner, die nach F₁-Kreuzung unserer Opaque 2-Linien mit Normallinien ausgelesen wurden, 45, 50 und sogar 74% mehr Lysin im ganzen Korn als normale durchsichtige Körner desselben Maiskolbens enthalten. Der Lysingehalt der undurchsichtigen Körner aus diesen Kreuzungen entspricht annähernd dem der Opaque 2-Elternlinien.Einige unserer Opaque 2-Linien enthalten 63 bis 122% mehr Lysin als die untersuchten normalen Inzuchtlinien. Im Vergleich zu Opaque 2 — Purdue weisen unsere 4 Opaque 2-Linien einen etwa gleichen Lysingehalt auf, während die SP-1 No. 15 25% mehr Lysin und 73% Tryptophan enthält. Unsere Opaque 2-Linien zeigen im ganzen Korn 55 bis 100% mehr Tryptophan als Opaque 2 — Purdue.Im Unterschied zum Lysingehalt zeigt die Übertragung des Tryptophangehalts von Opaque 2-Linien auf undurchsichtige Körner bei den Kreuzungen Variabilität.Wir fanden eine schwach positive Korrelation (r = + 0,3057) zwischen dem Lysin- und Tryptophangehalt bei den Opaque 2-Köinern.Unsere Opaque 2-Linien hatten mehr Lysin im Endosperm als Opaque 2-Purdue, und zwar 22,1 bis 36,6% bzw. 109,5 bis 142,6% im Vergleich zu den normalen Linien.Offensichtlich ist eine neue Quelle für das mutante Opaque 2-Gen in einer ganz neuen, genetisch unterschiedlichen Synthese einer großen Anzahl von Populationen entdeckt worden. Die neue Quelle hat dieselben genetischen und biochemischen Merkmale wie die Opaque 2-Mutante, die von Mertz, Bates und Nelson (1964) aufgefunden wurde.

     

Mitochondrial enzyme activities as biochemical markers of aging

The decrease of neurological performance in normal aging is directly related to brain oxidative stress and inversely related to lifespan. Male mice lifespan was increased by 8-10% (median and maximal lifespan, respectively) in mice with high spontaneous neurological activity, by 21-15% after moderate exercise; and by 25-20% after supplementation with vitamin E. Oxidative stress markers, TBARS and protein carbonyl content, were found increased on aging; a higher content of oxidation products is considered an effective aging factor, specially in the brain, with a majority of postmitotic cells. Mitochondrial enzyme activities, mitochondrial nitric oxide synthase (mtNOS), NADH dehydrogenase and cytochrome oxidase, behaved as markers of brain aging. The decrease in enzyme activities was directly related to the content of oxidation products and to the loss of neurological function in aged mice, this latter was determined in the tighrope and the T-maze tests. The above mentioned conditions that increased mice lifespan were effective to decrease the level of oxidative stress markers, and to retard the decreases in mitochondrial enzyme activities and neurological function associated to aging. The activities of mtNOS, NADH dehydrogenase and cytochrome oxidase may be used as indicators of the effectiveness of antiaging treatments.     

Self-splicing introns as a source for transposable genetic elements

Previous theories have suggested that some introns with the ability to self-splice are derived from transposable elements. However, an interpretation is given here that suggests retrotransposons and retroviruses (transposable elements which move via RNA intermediates) have evolved from self-splicing introns. This is based on the involvement of RNA intermediates, the ancestral nature of the self-splicing reaction, and the assumed presence of introns in an RNA world. Conserved sequences within the introns, essential for splicing, and their wide phylogenetic distribution also make it unlikely that they are descended from transposable elements. Mitochondrial plasmids of Neurospora species containing features of both introns and retrotransposons have a central role in the resolution of the problem and are considered here to support the view that introns are, or have been, sources of mobile elements. The possibility of other transposable elements arising from introns is also considered.     

Evaluation of saliva as a source of human DNA for population and association studies

A simple noninvasive procedure for saliva sample collection and DNA extraction was developed. On average, the amount of human DNA (as measured by a TaqMan-based assay) was about 11.4 microg/mL saliva, which is more than can be obtained from other noninvasive samples such as cheek swabs. However, the presence of large amounts of nonhuman DNA (up to 90% of the total extracted DNA) in saliva samples does necessitate DNA quantitation methods that are specific for human DNA. We were able to reliably and accurately type different genetic markers (mDNA sequences, Y-chromosomal single-nucleotide polymorphisms, and autosomal microsatellite loci) from saliva samples stored for up to 30 days at 37 degrees C, making this method well-suited for field conditions and convenient transportation of samples back to the laboratory. Thus, saliva can be considered a reliable source of DNA for a wide variety of genetic studies.     

Pangenesis as a source of new genetic information. The history of a now disproven theory.

Evolution is based on natural selection of existing biological phenotypic traits. Natural selection can only eliminate traits. It cannot create new ones, requiring a theory to explain the origin of new genetic information. The theory of pangenesis was a major attempt to explain the source of new genetic information required to produce phenotypic variety. This theory, advocated by Darwin as the main source of genetic variety, has now been empirically disproved. It is currently a theory mainly of interest to science historians.     

Human occupancy as a source of indoor airborne bacteria

Exposure to specific airborne bacteria indoors is linked to infectious and noninfectious adverse health outcomes. However, the sources and origins of bacteria suspended in indoor air are not well understood. This study presents evidence for elevated concentrations of indoor airborne bacteria due to human occupancy, and investigates the sources of these bacteria. Samples were collected in a university classroom while occupied and when vacant. The total particle mass concentration, bacterial genome concentration, and bacterial phylogenetic populations were characterized in indoor, outdoor, and ventilation duct supply air, as well as in the dust of ventilation system filters and in floor dust. Occupancy increased the total aerosol mass and bacterial genome concentration in indoor air PM(10) and PM(2.5) size fractions, with an increase of nearly two orders of magnitude in airborne bacterial genome concentration in PM(10). On a per mass basis, floor dust was enriched in bacterial genomes compared to airborne particles. Quantitative comparisons between bacterial populations in indoor air and potential sources suggest that resuspended floor dust is an important contributor to bacterial aerosol populations during occupancy. Experiments that controlled for resuspension from the floor implies that direct human shedding may also significantly impact the concentration of indoor airborne particles. The high content of bacteria specific to the skin, nostrils, and hair of humans found in indoor air and in floor dust indicates that floors are an important reservoir of human-associated bacteria, and that the direct particle shedding of desquamated skin cells and their subsequent resuspension strongly influenced the airborne bacteria population structure in this human-occupied environment. Inhalation exposure to microbes shed by other current or previous human occupants may occur in communal indoor environments.     

Proinflammatory factors in saliva as possible markers for periodontal disease

Studies have indicated that host inflammatory proteins, enzymes and indicators of bone metabolism present in saliva differ in different types of periodontal disease. However, the number of markers analyzed was limited and the effect of edentulousness was not examined. We measured the concentration of host inflammatory proteins: C-reactive protein (CRP), C3 and C4 complement components, alpha-2-macroglobulin (alpha-2M) and tumor-necrosis factor (TNF) in unstimulated saliva of 14 periodontally healthy (PH), 9 edentulous persons (EP), 10 patients with chronic periodontitis (CP) and 18 with aggressive periodontitis (AgP). TNF was below the level of detection in all samples except one. Edentulous persons and patients with CP had significantly reduced concentrations of CRP, C3 and alpha-2M. Edentulous persons and AgP patients had lower C4 concentrations. We can conclude that edentulous persons and CP patients have reduced salivary concentrations of host inflammatory proteins. These findings suggest that a reduction in host responsiveness might play a role in the pathogenesis of CP.     

Molecular markers and protein quantities as genetic descriptors in maize. I. Genetic diversity among 21 inbred lines

Twenty-one maize (Zea mays L.) inbred lines were analysed using isozyme electrophoresis, restriction fragment length polymorphism (RFLP), and two-dimensional electrophoresis of denatured proteins (2-D PAGE). Our goal was (1) to assess the genetic variability among these lines which are potential progenitors for the development of forage maize hybrids in Europe, and (2) to compare the relationship pattern revealed by the polymorphism at marker loci with the one derived from the amount of protein variability assessed by computer-assisted analysis of the 2-D electrophoregrams. Fourteen markers were obtained from isozyme polymorphism, 84 from the restriction fragment length polymorphism, and 70 from protein shifts revealed by 2-D PAGE. The Rogers' distance computed on the set of molecular markers was the most efficient to describe the pedigree relationships between lines. Quantitative protein data gave a picture of relationships between lines clearly different from the monogenic markers. When unrelated pairs of lines were considered, the Rogers' distance was weakly correlated to distances based on quantitative variations in the amount of protein which may be consistent with their polygenic control and the occurrence of gene interactions.