Linkage of PGK1 to X-linked severe combined immunodeficiency (IMD4) allows predictive testing in families with no surviving male

Summary We present a linkage map of DNA probes around the X-linked severe combined immunodeficiency (IMD4) locus at Xq11-13. DXS159 and PGK1 show no cross-overs with the disease locus (Lod 3.01 at = 0.00). The order of loci is DXS1-DXS106-(DXS159-PGK1-IMD4)-DXS72-DXYS1. Members of families whose carrier status has been established by X-inactivation patterns were included in the analysis. As the probe (pSPT/PGK), which is used for investigation of X-inactivation patterns, has been shown to be linked to the disease itself, it is possible to assign phase in mothers of sporadic cases who have been shown to be carriers, even when they have no surviving male offspring.     

PCNA-dependent DNA polymerase delta from rabbit bone marrow.

Proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta were partially purified and characterized from rabbit bone marrow. Rabbit DNA polymerase delta sediments at 8.2 S upon glycerol density gradient centrifugation. Similar to calf thymus PCNA-dependent DNA polymerase delta, a 125-123-kDa doublet and 48-kDa polypeptides correlate with DNA polymerase activity. Western blotting of rabbit DNA polymerase delta with polyclonal antibody to calf thymus PCNA-dependent DNA polymerase delta gives the same results as calf thymus delta; the 125-123-kDa doublet is recognized. PCNA-dependent DNA polymerase delta is resistant to inhibition by dideoxynucleotides and is relatively insensitive to inhibition by N2-[p-(n-butyl)phenyl]dGTP. A 3'-->5' exonuclease copurifies with the DNA polymerase. The processivity of DNA polymerase delta alone is very low but greatly increases with the addition of PCNA from rabbit bone marrow or calf thymus. Comparative studies of the original DNA polymerase delta from rabbit bone marrow demonstrate a lack of recognition by antibodies to calf thymus delta and a high degree of processivity in the absence of PCNA. Additionally, the originally described DNA polymerase delta is a single polypeptide of 122 kDa. These features would recategorize the original delta to the epsilon category by recently proposed convention. PCNA-dependent DNA polymerase delta is a relatively minor component of rabbit bone marrow compared to DNA polymerase alpha and PCNA-independent DNA polymerase delta (epsilon), the relative proportions being alpha, 60%; delta, 7%; and epsilon, 30%.     

Predation on Protozoa: its importance to zooplankton

Protozoa are an important component of both the nano- and microplanktonin marine and freshwater environments and are preyed upon byzooplankton, including suspension-feeding cope pods, some gelatinouszoopiankters and some first-feeding fish larvae. The clearancerates of suspension-feeding zooplankton for ciliates, in particular,are higher than for most phytoplankton. For at least some suspension-feedingzooplankton, protozoans are calculated to be quantitativelyan important component of the diet during certain seasons. Inlaboratory studies, protozoan components in the diet appearto enhance growth and survival of certain life-history stagesor enhance fecundity. These data suggest that protozoans arequalitatively as well as quantitatively important in the dietsof marine zooplankton. Most studies of predation on Protozoahave focused on the euphotic zone in nearshore waters. Predationon Protozoa is expected, however, to be particularly importantboth quantitatively and qualitatively in marine environmentsand seasons in which primary production is dominated by cells<5 µm in size, such as nearshore environments afterthe spring phytoplankton bloom, in oligotrophic waters, andin environments dominated by detritus-dominated food webs, suchas the deep sea. In detritus-dominated food webs, Protozoa maybe a source of essential nutrients and may thus facilitate utilizationof bacterial and detrital carbon by metazoan plankton.     

The biology and morphology of the marine harpacticoid copepod Heteropsyllus nunni Coull, during encystment diapause

Heteropsyllus nunni Coull, a meiobenthic harpacticoid copepod is the marine crustacean to undergo a state of diapause within a cyst. A 12 month field study indicated H. nunni adults reached peak population densities in winter, with nauplii maturing in the spring, becoming adults by April or May.At the last stage of development, a mature but unmated adult, they begin to prepare for encystment diapause. The copepods remain within their cyst in a state of diapause for 3–4 months during the summer only. Studies on the effects of temperature and photoperiod suggested that these two environmental cues are not crucial for induction or termination of diapause. Low temperature delayed development and time to encystment, while high temperatures accelerated development, making the time to encystment shorter. There were males than females in the cysts in laboratory experiments. Upon excystment, the copepods mate, and females begin egg production within one week. Adults that have excysted and mated die after a few weeks of active reproductive effort. Nauplii go on to mature and begin the univoltine diapause/reproductive cycle.The copepods prepare for dormancy in two ways: they begin to produce and store two types of secretory products to be used in cyst construction; then they produce large quantities of lipid to be used as a nutrient supply throughout diapause. Histochemistry of the cyst-building material indicated the lower urosome is full of two chemically different products. Dorsally, there is a storage sac of proteinaceous material. The ventral sac of secretory product is a mucopolysaccharide. The copepod builds the spherical cysts in a matrix of small and large sand grains. The cysts fit tightly around the ventral portion of the animal in the its flexed position: however, there is a large space between the cyst and the sides of the copepod.Biochemical analysis of the cyst showed it is composed of an amino acid complex similar to collagenous material. Scanning electron microscopy revealed a complex of large cuticular pores located in the lower urosome and caudal rami. There are specific pores for secretion of the two cyst-building products.     

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Uptake and transformation of metals and metalloids by microbial mats and their use in bioremediation

Summary Constructed microbial mats, used for studies on the removal and transformation of metals and metalloids, are made by combining cyanobacteria inoculum with a sediment inoculum from a metal-contaminated site. These mats are a heterotrophic and autotrophic community dominated by cyanobacteria and held together by slimy secretions produced by various microbial groups. When contaminated water containing high concentrations of metals is passed over microbial mats immobilized on glass wool, there is rapid removal of the metals from the water. The mats are tolerant of high concentrations of toxic metals and metalloids, such as cadmium, lead, chromium, selenium and arsenic (up to 350 mg L^–1). This tolerance may be due to a number of mechanisms at the molecular, cellular and community levels. Management of toxic metals by the mats is related to deposition of metal compounds outside the cell surfaces as well as chemical modification of the aqueous environment surrounding the mats. The location of metal deposition is determined by factors such as redox gradients, cell surface micro-environments and secretion of extra-cellular bioflocculents. Metal-binding flocculents (polyanionic polysaccharides) are produced in large quantities by the cyanobacterial component of the mat. Steep gradients of redox and oxygen exist from the surface through the laminated strata of microbes. These are produced by photosynthetic oxygen production at the surface and heterotrophic consumption in the deeper regions. Additionally, sulfur-reducing bacteria colonize the lower strata, removing and utilizing the reducing H₂S, rather than water, for photosynthesis. Thus, depending on the chemical character of the microzone of the mat, the sequestered metals or metalloids can be oxidized, reduced and precipitated as sulfides or oxides. For example precipitates of red amorphous elemental selenium were identified in mats exposed to selenate (Se-VI) and insoluble precipitates of manganese, chromium, cadmium, cobalt, and lead were found in mats exposed to soluble salts of these metals. Constructed microbial mats offer several advantages for use in the bioremediation of metal-contaminated sites. These include low cost, durability, ability to function in both fresh and salt water, tolerance to high concentrations of metals and metalloids and the unique capacity of mats to form associations with new microbial species. Thus one or several desired microbial species might be integrated into mats in order to design the community for specific bioremediation applications.