The complete nucleotide sequence of a 6,851-base pair (bp) member of the L1Md repetitive family from a selected random isolate of the BALB/c mouse genome is reported here. Five kilobases of the element contains two overlapping reading frames of 1,137 and 3,900 bp. The entire 3,900-bp frame and the 3' 600 bp of the 1,137-bp frame, when compared with a composite consensus primate L1 sequence, show a ratio of replacement to silent site differences characteristic of protein coding sequences. This more closely defines the protein coding capacity of this repetitive family, which was previously shown to possess a large open reading frame of undetermined extent. The relative organization of the 1,137- and 3,900-bp reading frames, which overlap by 14 bp, bears resemblance to protein-coding, mobile genetic elements. Homology can be found between the amino acid sequence of the 3,900-bp frame and selected domains of several reverse transcriptases. The 5' ends of the two L1Md elements described in this report have multiple copies, 4 2/3 copies and 1 2/3 copy, of a 208-bp direct tandem repeat. The sequence of this 208-bp element differs from the sequence of a previously defined 5' end for an L1Md element, indicating that there are at least two different 5' end motifs for L1Md.
To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from -340 to -481 bases upstream of the GluR2 AUG codon. The relative use of 5' start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold (+/- 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substitution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) elements within a GC-rich proximal GluR2 promoter region. The GluR2 silencer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfection of REST into neurons reduced GluR2 promoter activity in a silencer-dependent manner. Substitution of the GluR2 silencer by the homologous NaII RE1 silencer further reduced GluR2 promoter activity in non-neuronal cells by 30-47%. Maximal positive GluR2 promoter activity required both Sp1 and NRF-1 cis elements and an interelement nucleotide bridge sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region.
We have characterized a large repetitive element which has been found at seven different locations within the beta globin locus of the BALB/c mouse. This repeat has an unusual structure in that each of the different members has the same end of the element conserved while the other end terminates at a different point in each repeat member. The sequences within the repeats from the beta globin locus have homology with other repetitive families such as the MIF-1, Bam-5, R, and the BamHl families. These were recently proposed (T. Fanning, (1983) Nucleic Acids Res. 11, 5073-5091) to be part of a structure with the same organization which we found in the globin locus. Probing plaques from a BALB/c genomic library with sequences derived from the repeats in the globin locus shows that virtually all of the repeats from this family are organized in a manner consistent with the proposed structure.
FK1, a ferret ventricular full-length cDNA clone, encodes a 654-amino acid protein with 98% identity to human K+ transient outward current (Ito)-like HK1 (Tamkun et al. FASEB J.5: 331-337, 1991). FK1 is detectable in ferret brain, atrium, left and right ventricle, and kidney but not in skeletal muscle, endothelial cells, aorta, and liver. In Xenopus oocytes, FK1 cRNA gives rise to a rapidly activating and inactivating Ito-like current, which is highly K+ selective (Na(+)-to-K+ permeability ratio = 0.003). Activation occurs over an approximately 50-mV range (-40 to +10 mV) and displays a sigmoid delay in onset with potential-dependent time constants that decrease with depolarization. Steady-state activation can be described with either a simple Boltzmann relationship [half-activation potential (V1/2) = -25 mV, slope (k) = 10 mV] or a Boltzmann relationship raised to either the third or fourth power (alpha 3: V1/2 = -43 mV, kappa = 13.1 mV; alpha 4: V1/2 = -48 mV, kappa = 13.6 mV, where alpha is the activation variable). Inactivation kinetics are biexponential, with the main fast time constant becoming independent of membrane potential depolarized to 0 mV. Steady-state inactivation can be described with a single Boltzmann relationship (V1/2 = -57 mV, kappa = 5.0 mV). Fast inactivation is removed by NH2-terminal deletions. Recovery from inactivation (-90 mV) is quite slow (half-time = 4.8 +/- 2.5 s). In 2 mM extracellular K+ concentration ([K+]o), FK1 tail currents display conventional deactivation behavior; however, in 98 mM [K+]o the tail currents display "reopening" behavior. These results suggest a molecular basis for the electrophysiological similarities between ferret and human ventricular Ito (Campbell et al. J. Gen. Physiol. 101: 571-601, 1993; Näbauer et al. Circ. Res. 73: 386-394, 1993).
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