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B K Kay

Publications and source records attributed to B K Kay.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification and characterization of Src SH3 ligands from phage-displayed random peptide libraries.

We have used the Src homology 3 (SH3) domain to screen two phage-displayed random peptide libraries, each containing 2 x 10(8) unique members, and have identified a series of high affinity peptide ligands. The peptides possess similar proline-rich regions, which yield a consensus Src SH3-binding motif of RPLPPLP. We have confirmed this motif by screening a phage-displayed peptide library biased for SH3 ligands and identifying the same consensus sequence. Binding studies using synthetic peptides suggest that the RPLPPLP motif is important for SH3 binding and confers specificity for the Src SH3 domain, and that residues which flank the motif may also contribute to binding. Peptides that contain the RPLPPLP motif compete Src, but not Abl or phospholipase C gamma, SH3 interactions with SH3-binding proteins from cell lysates (IC50 = 1-5 microM). Furthermore, RPLPPLP-related peptides are able to accelerate progesterone-induced maturation of Xenopus laevis oocytes. A similar acceleration has been observed in oocytes treated with activated, but not normal, Xenopus Src, suggesting the possibility that the peptides are able to antagonize the negative regulation of Src activity by Src SH3 in vivo.

3T3 Cells↗
PubMed1994

Molecular basis of cardiac troponin T isoform heterogeneity in rabbit heart.

In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1 through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5' half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3' half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets.

We have examined the potential of isolating novel ligands from a library of M13 pIII-fusion phage displaying peptides composed of 38 random amino acids (aa). The library was panned with streptavidin (SA) and a polyclonal goat antimouse IgG Fc antibody (Ab) preparation coupled to paramagnetic beads. SA selected two classes of phage from the library. One class exhibited the aa motif, HP(Q/M) theta (where theta signifies a non-polar aa), similar to the motif identified by Devlin et al. [Science 249 (1990) 404-406] using a 15-aa random peptide library displayed on phage. The other class of phage had no discernible motif. In binding experiments, the non-HP(Q/M) theta phage had a slightly higher affinity for SA than did the motif phage. Both classes of SA-binding phage failed to bind native and non-glycosylated forms of avidin, even though SA and avidin are structurally similar and both proteins possess extraordinary affinities for biotin. The polyclonal goat anti-mouse IgG Fc Ab preparation selected phage displaying sequences similar to a region of the mouse IgG Fc. Thus, a single immunodominant epitope on the mouse IgG Fc was identified. Furthermore, a second phage displaying peptides with no discernible sequence similarities to mouse IgG Fc was isolated. Thus, an M13 library displaying 38-aa peptides can yield phage with affinity for various targets. Finally, we have observed a biological bias against odd numbers of Cys residues in the displayed peptides.

Amino Acid Sequence↗
PubMed1992

A two-motif isoform of the major calcium channel subunit in skeletal muscle.

Evidence is presented that two isoforms of the voltage-dependent, dihydropyridine-sensitive calcium channel alpha 1 subunit are present in newborn and adult skeletal muscle and that expression of these isoforms is developmentally regulated. A voltage-dependent calcium channel alpha 1 cDNA from newborn muscle was cloned and found to be identical to that published from the adult, except that it was 2 kb shorter owing to an internal deletion. Nucleotide sequences, Northern blots, reverse-transcriptase PCR experiments, and sequencing of the PCR product confirmed that a segment corresponding to the inner two repeats of the structural prototype four homologous motifs is missing from the immature isoform. Immunological studies using antisera raised against synthetic peptides that correspond to sequences in the two isoforms show that the abbreviated transcript is predominant in newborn muscle, whereas the four-repeat isoform is the major species in the adult.

Aging↗
PubMed1992

Multipurpose vectors for peptide expression on the M13 viral surface.

We have developed a set of three cloning vectors for the expression of polypeptides on the surface of the M13 viral coat. The M13mp8 genome has been engineered for expression of foreign protein sequences near the NH2-terminus of the mature pIII protein, which is present in five copies on the outside of each M13 viral particle. All three of the vectors carry the same two useful restriction sites for directed cloning of inserts in the pIII coding region; in addition, one vector carries the bacterial gene conferring resistance to the antibiotic tetracycline, and another expresses the lacZ' polypeptide that allows functional complementation of beta-galactosidase activity within the host bacterial cell. All of these vectors propagate well in E. coli DH5 alpha F' cells and do not require helper phage. We demonstrate that a bacteriophage, expressing an eleven amino acid epitope (from human c-myc) at the NH2-terminus of pIII in one of our vectors, can be purified from a vast mixture of other M13 phage through panning techniques. In particular, we find that the c-myc-expressing viral particles can be easily recovered from phage mixtures with the biotinylated form of the monoclonal antibody, 9E10, and streptavidin-coated MagneSphere beads.

Amino Acid Sequence↗
PubMed1991

Neural regulation of calmodulin in adult Xenopus leg muscle.

Two Ca(2+)-binding proteins important in regulating muscle responses to Ca2+ flux are differentially expressed following denervation of Xenopus laevis gastrocnemius. Levels of parvalbumin (PV) RNA transcripts and proteins decrease in abundance, while calmodulin (CaM) transcript and protein levels increase. Our studies on PV kinetics in Xenopus follow a pattern observed in other species, however, our observation of a concomitant increase in CaM has not been documented in any system. Molecular analyses of the Xenopus CaM gene indicate that its structure and upstream sequences are highly conserved across several vertebrate species and implicate several transcription factors in the regulation of its expression.

Amino Acid Sequence↗
PubMed1990

Parvalbumin expression in normal and mutant Xenopus embryos.

The levels and cellular distribution of PV were analyzed at the molecular level in normal and mutant Xenopus laevis embryos. In developing tadpoles, PV protein expression was restricted to fast-twitch myotomal cells, as shown by immunofluorescence experiments with myosin isoform-specific monoclonal antibodies. Both PV expressing and non-expressing myotomal cells can be cultured in vitro without neurons, and non-expressing cells have been shown to lack hybridizable levels of PV mRNA. In a paralyzed mutant, unresponsive, the level of PV protein was normal, but its cellular distribution was not. Thus, while neural input may not be necessary for PV expression in embryonic cells, one gene sequence appears needed for proper cellular distribution of PV in tadpole tail muscles.

Animals↗
PubMed1990

Talin and vinculin in the oocytes, eggs, and early embryos of Xenopus laevis: a developmentally regulated change in distribution.

We have investigated the expression and distribution of talin and vinculin in the oocytes, eggs, and embryos of Xenopus laevis. Antibodies to the previously characterized avian proteins stain several different Xenopus cell types identically by immunofluorescence: adhesion plaques of cultured kidney (A6) cells, the cell peripheries of oviduct cells, and the postsynaptic neuromuscular junctions of tadpole tail muscle fibers. These antibodies also identify cognate proteins of the appropriate sizes on immunoblots of A6 cell and oviduct lysates. Using these antibodies on ovarian tissue, we find talin to be highly localized at the cortices of oocytes and vinculin to be in the oocyte cytoplasm and absent from the oocyte cortex. In the cells of the ovarian layers that surround the oocytes, talin and vinculin can be detected as soluble and cytoskeletal components. Vinculin is first detectable as a cytoskeletal component in eggs, appearing some time during or between oocyte maturation and oviposition. During early embryo development, talin and vinculin are colocalized in the cortex of cleavage furrows and blastomeres. Thus, Xenopus oocytes and eggs display different distributions of talin and vinculin. The change from unlinked localization to colocalization appears to be developmentally regulated, occurring during the transition from oocyte to egg.

Animals↗
PubMed1990

Gene activation is required for developmentally programmed cell death.

The intersegmental muscles of the tobacco hawkmoth Manduca sexta die during the 36-hr period after metamorphosis. The trigger for cell death is a fall in the ecdysteroid titer. Commitment of the intersegmental muscles to degenerate involves selective repression and activation of ecdysteroid-responsive genes. When the pattern of gene expression is altered after injection of either 20-hydroxyecdysone or actinomycin D, the muscles persist. cDNA clones have been isolated for four genes that become abundantly expressed coincident with the commitment to degenerate. The data presented here indicate that programmed cell death is not due to the cessation of macromolecular synthesis in condemned cells but rather is due to the activation of a differentiative pathway.

Aging↗
PubMed1990

Potential for two isoforms of the A1 ribonucleoprotein in Xenopus laevis.

We have identified the Xenopus cognates for the RNA-binding protein A1. This protein has previously been shown to be one of the components of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex in rat and human cells. We have isolated several Xenopus clones from oocyte, tailbud embryo, and leg muscle cDNA libraries and determined their nucleotide sequences. Potentially, two different A1 isoforms are expressed in Xenopus; they have been termed XA1a and XA1b. Besides insertions and deletions, the XA1a protein sequence is 92% identical to the rat protein and suggests very similar secondary structures. There are two segments in the COOH-terminal domain where deletions or insertions are apparent: the rat protein does not have a 48-residue sequence that is present in the frog protein, and the frog protein does not have a 12-residue sequence that is present in the rat protein. We have confirmed that the XA1a protein is larger than rat A1 by in vitro transcription, translation, and gel electrophoresis. The second isoform, XA1b, is very similar to the XA1a isoform, except it has a different COOH terminus due to the absence of a 73-nucleotide region from its cDNA clones. Transcripts representing both isoforms have been detected in various Xenopus RNA preparations by polymerase chain reaction experiments with A1-specific oligonucleotides. Our findings suggest that the isoforms are encoded by one or two genes and are the result of alternative splicing. We discuss the biological implications of having two forms of the A1 component of hnRNP particles.

Amino Acid Sequence↗
PubMed1988

Differential expression of the Ca2+-binding protein parvalbumin during myogenesis in Xenopus laevis.

We have used immunocytological techniques to examine the developmental expression of the Ca2+-binding protein parvalbumin in Xenopus laevis embryos. Western blot experiments show that at least three different forms of parvalbumin are expressed during embryogenesis; the tadpole tail expresses one form, adult brain expresses another, mylohyoid muscle expresses both, and gastrocnemius and sartorius muscles express these two plus a third form. Parvalbumin (PV) is first detectable by immunofluorescence at stages 24-25 of development, a time when myotomal muscles are differentiating and contractile activity occurs spontaneously in embryos. At metamorphosis, PV is expressed in developing limb muscles. While the majority of skeletal muscle fibers express high levels of PV in both embryos and adults, a second fiber type has no detectable PV. The arrangement of PV-containing fibers is stereotyped in each muscle group examined. Histochemical staining of tadpole muscles indicate that PV-containing fibers correspond to fast-twitch skeletal muscles, whereas those without PV correspond to slow-twitch muscles. During tail resorption at metamorphosis, PV appears to be extruded from dying tail muscle cells and taken up by phagocytic cells.

Animals↗
PubMed1988

Patterns of N-CAM expression during myogenesis in Xenopus laevis.

The neural cell adhesion molecule (N-CAM) is seen in the membrane of nerves and muscles from several vertebrate species. Using indirect immunofluorescence, we have examined the expression of this protein during embryonic and postembryonic myogenesis in the African clawed frog, Xenopus laevis. While good staining for N-CAM was seen in neuronal tissues at all stages examined, no staining of embryonic muscle was observed, including both mononucleated and polynucleated myoblasts. In contrast, limb muscles formed at metamorphosis showed strong expression of N-CAM. The developing limb muscles eventually lose their N-CAM, but will reexpress it dramatically when denervated. These observations suggest that myogenesis programs executed at different stages of development can display distinct patterns of N-CAM expression.

Animals↗
PubMed1987

Expression of the Ca2+-binding protein, parvalbumin, during embryonic development of the frog, Xenopus laevis.

A cDNA segment encoding the Ca2+-binding protein, parvalbumin, was isolated with the use of antibodies, from a lambda gtll expression library of Xenopus laevis tadpole poly(A)+ RNAs. The bacterially expressed beta-galactosidase-parvalbumin fusion protein of one lambda recombinant shows high affinity 45Ca2+ binding. The sequence of the tadpole parvalbumin is highly similar to previously characterized beta-parvalbumins of other organisms. Data from protein and RNA blotting experiments demonstrate that parvalbumin is absent in oocytes, eggs, and early staged embryos, and only becomes expressed during embryogenesis at the time of myogenesis. The protein can be detected in individual developing muscle cells and in muscle fibers of tadpole tail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos.

Amino Acid Sequence↗
PubMed1984

Transcription of a long, interspersed, highly repeated DNA element in Xenopus laevis.

We have analyzed the transcription of 1723, a long, repeated DNA element that is interspersed in the genome of Xenopus laevis (B. K. Kay and I. B. Dawid (1983) J. Mol. Biol. 170, 583-596). We have detected RNA homologous to 1723 in total cellular RNA from ovaries, embryos, liver, and cultured kidney cells. Transcripts from both strands of the element are present at similar concentrations in these different RNA preparations. In oocytes, approximately 100 pairs of lampbrush chromosome loops are active in the transcription of 1723 elements. The abundance of 1723 RNA increases during embryogenesis, with the highest level reached at the tadpole stage. From cellular fractionation studies, we conclude that 1723 transcripts are largely limited to the nucleus.

Animals↗
PubMed1983

The 1723 element: a long, homogeneous, highly repeated DNA unit interspersed in the genome of Xenopus laevis.

We describe a highly repeated DNA element in the Xenopus laevis genome. This sequence, named the 1723 element, was first identified among sequences that are transcribed during embryonic development. The element is present in about 8500 copies per haploid genome, which together accounts for about 2.4% of the genome. Most copies of the element have highly conserved restriction maps, and are interspersed in the genome. The copies range in size from 6000 to 10,000 base-pairs due to an expandable region that contains variable numbers of a tandemly repeating 183 to 204 base-pair unit. The element is framed by an imperfect 18 base-pair inverted sequence, and inverted repeats of 180 to 185 base-pairs are nearby. Sequence analysis of DNA adjacent to three cloned elements shows that the elements are flanked by 8 base-pair direct repeats. These and other properties of 1723 suggest that it may be transposable.

Animals↗