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K L Bennett

Publications and source records attributed to K L Bennett.

42 records · Page 3Linked to original sources

Most highly repeated dispersed DNA families in the mouse genome.

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.

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Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species.

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The effect of stimulation of atrial receptors on the plasma concentration of vasopressin.

The effect of stimulation of the left atrial receptors on plasma concentration of vasopressin and urine flow was studied in dogs anaesthetized with chloralose. Distending a large balloon in the left atrium, so as to occlude the mitral valve and increase left atrial pressure, consistently resulted in a decrease in plasma vasopressin and an increase in urine flow. Discrete stimulation of the left atrial receptors, by distending small balloons in the pulmonary vein - left atrial junctions and in the left atrial appendage, consistently resulted in a decrease in plasma vasopressin and an increase in urine flow. The left atrial receptors were stimulated discretely in dogs in which bretylium tosylate, atropine and atenolol had been administered. In these dogs distension of the small balloons was not associated with significant changes in heart rate, left atrial pressure or arterial blood pressure and consistently resulted in a decrease in plasma vasopressin and an increase in urine flow. It is concluded that this decrease in the plasma concentration of vasopressin is a direct result of discrete stimulation of the atrial receptors and is not secondary to any haemodynamic changes. The exact role of this decrease in plasma concentrations of vasopressin in the diuretic response to stimulation of the left atrial receptors remains to be established.

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Use of repetitive DNA sequences to distinguish Mus musculus and Mus caroli cells by in situ hybridization.

Mammalian chimaeras have proved useful for investigating early steps in embryonic development. However, a complete clonal analysis of cell lineages has been limited by the lack of a marker which is ubiquitous and can distinguish parental cell types in situ. We have developed a cell marker system which fulfils these criteria. Chimaeric mice were successfully produced from two mouse species which possess sufficient genetic differences to allow unequivocal identification of parental cell types. DNA-DNA in situ hybridization with cloned, species-specific sequences was performed to distinguish the parental cell types. We have identified a cloned, Mus musculus satellite DNA sequence which shows hybridization differences between Mus musculus and Mus caroli DNA. This clone was used a a probe in in situ hybridizations to bone marrow chromosomes from Mus musculus, Mus caroli, and an interspecific F1 hybrid. The clone could qualitatively distinguish Mus musculus from Mus caroli chromosomes after in situ hybridization, even when they were derived from the same F1 hybrid cell. Quantitation of this hybridization to interphase nuclei from bone marrow spreads indicates that the probe can successfully distinguish Mus musculus from Mus caroli cells and can determine the percentage contribution of Mus musculus in mixtures of bone marrow cells of these species and in chimaeric bone marrow cell preparations.

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Mapping the structural genes coding for the major urinary proteins in the mouse: combined use of recombinant inbred strains and somatic cell hybrids.

We have mapped the multiple (15-25) genes coding for the hormonally regulated major urinary proteins (MUPs) of the mouse by using a cloned cDNA probe. By Southern blot analysis of DNA from Chinese hamster-mouse somatic cell hybrids, all of the MUP genes were found to be on chromosome 4. Different inbred mouse strains showed DNA polymorphism in their MUP Southern hybridization pattern. Analysis of recombinant inbred strains derived from these parent strains has shown that all the polymorphisms are linked to the MUP-a locus on chromosome 4. The combination of these mapping techniques should be applicable to many cloned DNA sequences.

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Rapid characterization of chemically-modified proteins by electrospray mass spectrometry.

Electrospray mass spectrometry (ESI-MS) has been used to examine monoclonal antibodies (MAbs), antibody fragments (Fab and Fc), modified fragments, and a range of other chemically-modified proteins as part of a study aimed at establishing ESI-MS as a method for the characterization of radioimmunoconjugates. This has been approached from two angles. Firstly, ESI-MS of complexes formed between chelators and other small molecules conjugated to hen egg white lysozyme (HEL) (14 kDa) demonstrate the considerable advantages of this powerful new technique compared with existing methods for the characterization of chemically-conjugated proteins. Molecular weights can be determined rapidly to within 0.01-0.05% and with good sensitivity (10-50 pmol total), thus providing specific structural information and opening the way for ESI-MS to be applied widely for the structural characterization of radioimmunoconjugates. Secondly, the conditions for ESI-MS of intact antibodies and antibody fragments have been examined in detail, and we have shown that the addition of up to 10 biotin molecules to the 50 kDa Fab fragment can be easily detected in ESI mass spectra, thus demonstrating the potential for the characterization of modified MAb fragments and metabolites. Finally, the strengths and limitations of ESI-MS of intact antibodies are discussed, and these results indicate that it may only be possible to detect average shifts in the mass of intact antibodies following modification.

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