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L Enquist

Publications and source records attributed to L Enquist.

At least 19 recordsLinked to original sources
PubMed2001

Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus.

We report the development of a pseudorabies virus that can be used for retrograde tracing from selected neurons. This virus encodes a green fluorescent protein marker and replicates only in neurons that express the Cre recombinase and in neurons in synaptic contact with the originally infected cells. The virus was injected into the arcuate nucleus of mice that express Cre only in those neurons that express neuropeptide Y or the leptin receptor. Sectioning of the brains revealed that these neurons receive inputs from neurons in other regions of the hypothalamus, as well as the amygdala, cortex, and other brain regions. These data suggest that higher cortical centers modulate leptin signaling in the hypothalamus. This method of neural tracing may prove useful in studies of other complex neural circuits.

Afferent Pathways↗
PubMed1987

Site-specific insertion of DNA into a pseudorabies virus vector.

A simple, efficient method for introducing recombinant DNA into a herpesvirus vector and retrieving it at a later time has been developed. By using the Cre-lox site-specific recombination system of coliphage P1, DNA can be readily inserted in vitro into a pseudorabies virus (PRV) vector containing the lox recombination site. The vector PRV42 contains a lox site within the nonessential gIII gene, which encodes a virion envelope glycoprotein. Incubation in vitro of PRV42 DNA with Cre protein and a circular plasmid containing a lox site generates approximately 5% recombinant molecules having the plasmid integrated into the PRV genome at the lox site. Transfection of the reaction mixture into cultured cells allows recovery of the infectious recombinant virus, which is readily identified by a nondestructive "black-plaque assay" using a gIII-specific monoclonal antibody. PRV42 plaques stain black when treated with the gIII monoclonal antibody and a peroxidase-linked second anti-antibody because the lox site placed within the gIII gene of PRV42 does not destroy the gIII epitope. However, Cre-mediated integration of heterologous DNA at the lox site disrupts the gIII epitope so that the resulting recombinant virus produces white plaques. The recombinant virus is infectious, stable, and grows as well as the parental PRV42 vector. The inserted plasmid can be efficiently excised (greater than 50%) from viral DNA by Cre and recovered by transformation of Escherichia coli.

Coliphages↗
PubMed1981

Downstream regulation of int gene expression by the b2 region in phage lambda.

Expression of the int gene after phage lambda infection normally requires the products of genes cII and cIII. However, when the phage carries a deletion in the nonessential b2 region adjacent to int, efficient synthesis of active Int protein does not require cII and cIII function. This inhibition of Int synthesis by nucleotide sequences downstream from the int structural gene behaves in a cis-dominant fashion in mixed infections. It is specific for PL- and not pI-initiated transcripts. Based on these observations, and those of others, a model is proposed in which Int translation from the pL transcript is inhibited by the interaction of downstream b2 nucleotide sequences and nucleotide sequences in the int region. The data imply a novel temporal mechanism regulating prophage lambda induction: circularization of the prophage genome results in the transposition of inhibitory b2 region sequences next to int and blocks further Int protein synthesis beyond the low level required for excision. As a consequence of this process, the control of int expression is transferred from the pL promoter to pI and the cII/cIII system. Such a genetic regulatory mechanism involving the rearrangement of genetic elements downstream from a structural gene may be of general use during development in other systems.

Bacteriophage lambda↗
PubMed1981

Identification of OmpR: a positive regulatory protein controlling expression of the major outer membrane matrix porin proteins of Escherichia coli K-12.

We report here on the cloning of a gene located within the ompB locus of E. coli K-12. This gene, designated ompR, resides on a 10.9-kilobase EcoRI fragment that we cloned, using a lambda vector and in vitro packaging techniques. By subcloning portions of this fragment into the high-copy-number plasmid pBR322, we have isolated the ompR gene on a 1.4-kilobase EcoRI-AvaI fragment. This fragment has been characterized physically and will facilitate a more detailed study of the role and mechanism of porin regulation by the ompB locus.

Bacterial Proteins↗
PubMed1979

A simple technique for the isolation of deletion mutants of phage lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.

Bacteriophage lambda↗
PubMed1979

Analysis of coliphage lambda mutations that affect Q gene activity: puq, byp, and nin5.

We describe in this paper the isolation and characterization of a class of mutations, designated puq, that allow phage lambda to grow better under conditions that limit the synthesis of the phage Q gene product. These mutations were located between phage genes P and Q, a region of the lambda chromosome containing two gene N-independent mutations, nin5 and byp, that we also show to be puq mutations. Whereas the puq-3 and puq-16 mutations probably map under the nin5 deletion, the byp mutation maps between this deletion and the Q lambda-Q phi 80 crossover point. These mutations likely act by increasing the synthesis of the Q gene product. We demonstrate that the clear-plaque phenotype and reduced lysogenization frequency of byp mutants depend on increased Q gene activity. The significance of these results in understanding how transcription proceeds through the P-Q region of the lambda genome is discussed.

Bacteriophage lambda↗
PubMed1977

EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from higher organisms: the lambdagtWES system.

A derivative of bacteriophage lambda has been modified and tested together with an appropriate host system to meet the criteria of EK2 biologic containment for cloning DNA from higher organisms. In this report certain of the safety features are summarized and some of the tests carried out to confirm the containment properties of the vector are described. The cloning efficiency of this system, together with available gene purification and hybrid screening technology, indicate that it can be used to clone DNA fragments carrying specific, unique mammalian genes.

Animals↗
PubMed1977

ColE1 cloning of a ribosomal RNA promoter region from lambdarifd18 by selection for lambda integration and excision functions.

The expression of the ribosomal RNA gene carried by the lambda transducing phage lambdarifd18 is shown to be subject to stringent amino acid control. lambdarifd18 DNA was digested with endonuclease EcoRI and ligated to similarly restricted ColE1 plasmid DNA. Selection for expression of lambda integration and excision gene activity carried by the same DNA fragment results in cloning of the promoter proximal portion of the 16S ribosomal RNA gene. The resulting chemera expresses lambda integration and excision functions as well as encoding the promoter proximal half of a 16S ribosomal RNA gene.

Bacteriocin Plasmids↗
PubMed1977

In vitro packaging of a lambda Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1.

In this report we describe a coliphage lambda vector system for cloning endo R. EcoRI DNA fragments. This system differs significantly from those previously described in two ways. First, restricted and ligated DNA is encapsidated in vitro. Second, with increasing lambda DNA size in the range 78 to 100% that of wild-type, the efficiency of DNA encapsidation into infectious phage particles markedly increases. For lambda wild-type DNA the efficiency of in vitro packaging (10(6) to 10(7) plaques produced per microgram of added DNA) is equal to, or better than, the standard CaCl2 transfection method. The use of a Dam mutation to facilitate recognition of size classes of inserted fragments is described. Using this vector and in vitro packaging, several E. coli and phage P1 and R.EcoRI fragments were cloned.

Coliphages↗