Horizontal gene transfer among genomes: The complexity hypothesis

Increasingly, studies of genes and genomes are indicating that considerable horizontal transfer has occurred between prokaryotes. Extensive horizontal transfer has occurred for operational genes (those involved in housekeeping), whereas informational genes (those involved in transcription, translation, and related processes) are seldomly horizontally transferred. Through phylogenetic analysis of six complete prokaryotic genomes and the identification of 312 sets of orthologous genes present in all six genomes, we tested two theories describing the temporal flow of horizontal transfer. We show that operational genes have been horizontally transferred continuously since the divergence of the prokaryotes, rather than having been exchanged in one, or a few, massive events that occurred early in the evolution of prokaryotes. In agreement with earlier studies, we found that differences in rates of evolution between operational and informational genes are minimal, suggesting that factors other than rate of evolution are responsible for the observed differences in horizontal transfer. We propose that a major factor in the more frequent horizontal transfer of operational genes is that informational genes are typically members of large, complex systems, whereas operational genes are not, thereby making horizontal transfer of informational gene products less probable (the complexity hypothesis).

Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.

Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo

Both caspase-1- and caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. We assessed the contributions of these caspases to Fas signaling in hepatocyte cell death in vitro. Although wild-type, caspase-1−/−, and caspase-3−/− hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3−/− hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1−/− apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation were seen within 6 hr, but neither event was observed for caspase-3−/− hepatocytes. We extended these studies to thymocytes and found that apoptotic caspase-3−/− thymocytes exhibited similar “abnormal” morphological changes and delayed DNA fragmentation observed in hepatocytes. Furthermore, the cleavage of various caspase substrates implicated in mediating apoptotic events, including gelsolin, fodrin, laminB, and DFF45/ICAD, was delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.

Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: Direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7×; LPL1, 1.8×), core cholesteryl ether (LPL2, 2.3×; LPL1, 2.7×), and apolipoprotein (LPL1, 1.8×; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.

Type 2 diabetes: Evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs

We are conducting a genome scan at an average resolution of 10 centimorgans (cM) for type 2 diabetes susceptibility genes in 716 affected sib pairs from 477 Finnish families. To date, our best evidence for linkage is on chromosome 20 with potentially separable peaks located on both the long and short arms. The unweighted multipoint maximum logarithm of odds score (MLS) was 3.08 on 20p (location, x̂ = 19.5 cM) under an additive model, whereas the weighted MLS was 2.06 on 20q (x̂ = 57 cM, recurrence risk, λ̂s = 1.25, P = 0.009). Weighted logarithm of odds scores of 2.00 (x̂ = 69.5 cM, P = 0.010) and 1.92 (x̂ = 18.5 cM, P = 0.013) were also observed. Ordered subset analyses based on sibships with extreme mean values of diabetes-related quantitative traits yielded sets of families who contributed disproportionately to the peaks. Two-hour glucose levels in offspring of diabetic individuals gave a MLS of 2.12 (P = 0.0018) at 9.5 cM. Evidence from this and other studies suggests at least two diabetes-susceptibility genes on chromosome 20. We have also screened the gene for maturity-onset diabetes of the young 1, hepatic nuclear factor 4-a (HNF-4α) in 64 affected sibships with evidence for high chromosomal sharing at its location on chromosome 20q. We found no evidence that sequence changes in this gene accounted for the linkage results we observed.

Genomic evidence for two functionally distinct gene classes

Analyses of complete genomes indicate that a massive prokaryotic gene transfer (or transfers) preceded the formation of the eukaryotic cell. In comparisons of the entire set of Methanococcus jannaschii genes with their orthologs from Escherichia coli, Synechocystis 6803, and the yeast Saccharomyces cerevisiae, it is shown that prokaryotic genomes consist of two different groups of genes. The deeper, diverging informational lineage codes for genes which function in translation, transcription, and replication, and also includes GTPases, vacuolar ATPase homologs, and most tRNA synthetases. The more recently diverging operational lineage codes for amino acid synthesis, the biosynthesis of cofactors, the cell envelope, energy metabolism, intermediary metabolism, fatty acid and phospholipid biosynthesis, nucleotide biosynthesis, and regulatory functions. In eukaryotes, the informational genes are most closely related to those of Methanococcus, whereas the majority of operational genes are most closely related to those of Escherichia, but some are closest to Methanococcus or to Synechocystis.

Mental chronometry using latency-resolved functional MRI

Vascular responses to neural activity are exploited as the basis of a number of brain imaging techniques. The vascular response is thought to be too slow to resolve the temporal sequence of events involved in cognitive tasks, and hence, imaging studies of mental chronometry have relied on techniques such as the evoked potential. Using rapid functional MRI (fMRI) of single trials of two simple behavioral tasks, we demonstrate that while the microvascular response to the onset of neural activity is delayed consistently by several seconds, the relative timing between the onset of the fMRI responses in different brain areas appears preserved. We examined a number of parameters that characterize the fMRI response and determined that its onset time is best defined by the inflection point from the resting baseline. We have found that fMRI onset latencies determined in this manner correlate well with independently measurable parameters of the tasks such as reaction time or stimulus presentation time and can be used to determine the origin of processing delays during cognitive or perceptual tasks with a temporal accuracy of tens of milliseconds and spatial resolution of millimeters.

Adenylyl cyclase 6 is selectively regulated by protein kinase A phosphorylation in a region involved in Gαs stimulation

Receptors activate adenylyl cyclases through the Gαs subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by Gαs. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of Gαs. Similar treatment of AC1 or AC2 did not affect Gαs stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of Gαs stimulation. A peptide encoding the region 660–682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of Gαs. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660–682 of AC6 is involved in regulation of signal transfer from Gαs. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.

Clinical outcome in relation to care in centres specialising in cystic fibrosis: cross sectional study

Objectives: To assess the effect on clinical outcome of managing paediatric and adult patients with cystic fibrosis at specialised cystic fibrosis centres.

ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma

DPC4 is known to mediate signals initiated by type β transforming growth factor (TGFβ) as well as by other TGFβ superfamily ligands such as activin and BMP (bone morphogenic proteins), but mutational surveys of such non-TGFβ receptors have been negative to date. Here we describe the gene structure and novel somatic mutations of the activin type I receptor, ACVR1B, in pancreatic cancer. ACVR1B has not been described previously as a mutated tumor-suppressor gene.

Biological clocks

Circadian rhythms describe biological phenomena that oscillate with an ≈24-hour cycle. These rhythms include blood pressure, body temperature, hormone levels, the number of immune cells in blood, and the sleep-wake cycle. In this paper, we will focus on common genes between species that are responsible for determining the circadian behavior, especially some transcription factors (i.e., switch genes) that serve to regulate many circadian rhythm genes. The intent of this summary is to introduce the common molecular mechanism of biological clocks between flies and humans and then to describe the research from three laboratories that was presented in the session.

Different modes of hippocampal plasticity in response to estrogen in young and aged female rats

Estrogen regulates hippocampal dendritic spine density and synapse number in an N-methyl-d-aspartate (NMDA) receptor-dependent manner, and these effects may be of particular importance in the context of age-related changes in endocrine status. We investigated estrogen's effects on axospinous synapse density and the synaptic distribution of the NMDA receptor subunit, NR1, within the context of aging. Although estrogen induced an increase in axospinous synapse density in young animals, it did not alter the synaptic representation of NR1, in that the amount of NR1 per synapse was equivalent across groups. Estrogen replacement in aged female rats failed to increase axospinous synapse density; however, estrogen up-regulated synaptic NR1 compared with aged animals with no estrogen. Therefore, the young and aged hippocampi react differently to estrogen replacement, with the aged animals unable to mount a plasticity response generating additional synapses, yet responsive to estrogen with respect to additional NMDA receptor content per synapse. These findings have important implications for estrogen replacement therapy in the context of aging.