Construction and Characterization of Deletion Mutations in Domain C of ARSl

115 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.Autonomously Replicating Sequences, or ARS elements, promote high-frequency transformation and extrachromosomal maintenance of plasmids in Saccharomyces cerevisiae, properties expected of DNA replication origins. A series of overlapping deletions in one flanking region (Domain C) of the ARSl element was constructed, and the effect of the deletions on the maintenance of various multicopy and single-copy plasmids examined. Analysis of the stabilities and copy numbers of multicopy plasmids indicated that while the core consensus element is absolutely required for extrachromosomal maintenance, the absence of Domain C has little effect. The loss rates of centromere (single-copy) plasmids increased slightly as the deletions approached the core consensus, suggesting that a block of sequence between 225 and 255 nucleotides from the consensus contains an element important to the maximal function of ARSl. These results also suggested that ARSl plays a role in replication, but has no effect on the segregation of centromere plasmids.Comparison of multicopy plasmids of different sizes, each with an intact ARSl element, indicated a destabilizing effect of sequences derived from the E. coli cloning vector pBR322. A small amount of pBR322 (about a kilobase) was tolerated, and the bacterial ori was not responsible for the destabilizing effect.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Increased glutamate receptor and transporter expression in the cerebral cortex and striatum of gcdh-/- mice: possible implications for the neuropathology of glutaric acidemia type I.

We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh-/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh-/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh-/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh-/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh-/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh-/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh-/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh-/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I

Glutamate transporter (GLT1 and GLAST) mRNA expression in cerebral cortex (A) and striatum (B) of wild type (WT) and <i>Gcdh-/-</i> mice submitted to a normal (0.9% lysine) or high lysine (4.7%) diet.

<p>A). Results are expressed as mean ± standard deviation for five independent experiments (animals) per group. * p≤0.05, **p≤0.01 compared to WT; #p≤0.05 compared to <i>Gcdh</i>^-/- mice submitted to a normal diet (Student's t test for unpaired samples).</p

Glutamate receptor mRNA expression levels in cerebral cortex and striatum from wild type (WT) and <i>Gcdh^-/-</i> mice at 7 (A), 30 (B) and 60 (C) days of life.

<p>Results are expressed as mean ± standard deviation for five independent experiments (animals) per group. *p≤0.05, **p≤0.01, ***p≤0.001 compared to WT (Student's t test for unpaired samples).</p

NMDA and non-NMDA receptor mRNA expression in cerebral cortex of 30-day-old wild type (WT) and <i>Gcdh^-/-</i> mice submitted to a normal (0.9% lysine) or high lysine (4.7%) diet.

<p>NR1 (A), NR2A (B), NR2B (C), GluR2 (D) and GluR6 (E). Results are expressed as mean ± standard deviation for five independent experiments (animals) per group. *p≤0.05 compared to WT; **p≤0.01, ***p≤0.001 compared to WT; ^#p≤0.01 compared to <i>Gcdh^-/-</i> mice submitted to a normal diet (Student's t test for unpaired samples).</p