Jeffery D. Fritz

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins myofibrillar proteins

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.

Cloning and chromosomal mapping of the murine norepinephrine transporter.

Abstract: The norepinephrine (NE) transporter (NET), a target of many clinically prescribed antidepressants, regulates noradrenergic neurotransmission by efficiently clearing NE from synaptic spaces after release. To advance our understanding of NET gene structure, regulation, and potential associations with complex behavioral trait loci, we amplified a mouse norepinephrine transporter (mNET) cDNA from placenta total RNA and utilized mNET probes to isolate and characterize the mNET gene. Inferred translation of the major open reading frame of the mNET cDNA predicts a 617‐amino acid protein with 12 putative membrane‐spanning regions and 94% identity to human NET. The coding exons of the mNET cDNA were found to be spread across >36 kb of 129/Svj genomic DNA, with exon‐intron boundaries bearing consensus gt/ag splice sites. Sequence upstream (202 bp) of the inferred translation initiation site matched the sequence of 5′ rapid amplification of cDNA ends products from brain mRNA with no evidence for intervening introns and is preceded by a TATA box and canonical transcriptional regulatory elements that may play a role in mNET expression in vivo. Probes derived from mNET cDNA identified species‐specific MspI restriction fragment length variations within the mNET gene that were utilized to position the gene (Slc6a5) to murine chromosome 8, one recombinant distal to D8Mit15. This site is within a recently defined quantitative trait locus defined for ethanol sensitivity in LSXSS recombinant inbred mice, Lore4. The status of Slc6a5 as a candidate gene for alcohol sensitivity is discussed with respect to studies noting ethanol‐induced alterations in brain NE receptors, NE receptor‐linked adenylate cyclase, and NE transport.

Expression of Deletion-Containing Dystrophins in mdx Muscle: Implications for Gene Therapy and Dystrophin Function

ABSTRACT: The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane. Only those constructs containing the carboxyl terminus were able to stabilize dystrophin-associated proteins (DAP) at the membrane, consistent with other studies that suggest that this domain is critical to DAP binding. Colocalization with DAP was not necessary for membrane localization of the various dystrophin molecules. However, stabilization and co-localization of the DAP did seem to be a prerequisite for expression and/or stabilization of mutant dystrophins beyond 1 wk and these same criteria seemed important for mitigating the histopathological consequences of dystrophin deficiency.

Partial titin cDNA sequence isolated from rabbit cardiac muscle RNA

SummaryTwo regions of the rabbit cardiac titin cDNA were amplified from rabbit cardiac muscle total RNA using primers based on rabbit skeletal muscle titin (connectin) cDNAs. These 1.7 kb and 1.5 kb RNA-PCR products were based on the 3′ regions of the skeletal muscle titin clones CE12 and MS2, respectively. The cDNA sequence of the 1.7 kb product was extended an additional 1.5 kb by a novel 3′ extension technique which used random primers in RNA-PCR. The cardiac titin cDNAs were 99% identical in nucleotide sequence to their skeletal muscle counterparts and predicted two types of 100-residue repeats, Southern blot analysis suggested that both cardiac and skeletal titin are encoded by the same gene. PCR amplification of human genomic DNA with titin specific primers indicated that there is strong sequence similarity between rabbit and human titin sequences. The successful amplification of a 907 basepair region from human genomic DNA suggested that titin contains large exons which span multiple motif borders. This may be particularly advantageous in the processing of such a large RNA transcript.

Gene-Based Neurotransmitter Modulation in Cerebellar Granule Neurons

Abstract: The human glutamic acid decarboxylase (GAD) gene was transferred into rat cerebellar granule neurons. Following adenoviral‐mediated gene transfer, nearly 100% of the neurons had transgene expression that persisted for the duration of their survival in culture. GABA levels were elevated both in the growth media and in lysates of GAD‐modified granule neurons. In GAD‐modified neurons, extracellular GABA levels steadily increased with time, whereas intracellular GABA levels peaked 10 days after gene transfer. GAD‐modified neurons released both glutamate and GABA into the surrounding media before and after potassium‐induced stimulation, but only the release of glutamate was sensitive to potassium stimulation. These data suggest that glutamatergic neurons, which initially contained no detectable GABA, can be genetically modified to release GABA constitutively.

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin

SummaryA 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3′ end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.

Characterization of a partial cDNA clone encoding porcine skeletal muscle titin: Comparison with rabbit and mouse skeletal muscle titin sequences

1. A cDNA fragment encoding 571 amino acid residues of porcine skeletal muscle titin was isolated from total RNA using RNA-PCR. 2. The porcine titin clone hybridized to a large RNA species (> 23 kb) in rabbit cardiac muscle, rabbit skeletal muscle and porcine skeletal muscle. 3. The porcine skeletal muscle titin clone encoded two types of 100-residue motifs and its amino acid sequence was 96% and 93% identical to the corresponding sequence of rabbit and mouse skeletal muscle titin, respectively. These results suggest that the titin sequence is highly conserved among mammalian species.