Ecological and evolutionary processes at expanding range margins.

Many animals are regarded as relatively sedentary and specialized in marginal parts of their geographical distributions. They are expected to be slow at colonizing new habitats. Despite this, the cool margins of many species' distributions have expanded rapidly in association with recent climate warming. We examined four insect species that have expanded their geographical ranges in Britain over the past 20 years. Here we report that two butterfly species have increased the variety of habitat types that they can colonize, and that two bush cricket species show increased fractions of longer-winged (dispersive) individuals in recently founded populations. Both ecological and evolutionary processes are probably responsible for these changes. Increased habitat breadth and dispersal tendencies have resulted in about 3- to 15-fold increases in expansion rates, allowing these insects to cross habitat disjunctions that would have represented major or complete barriers to dispersal before the expansions started. The emergence of dispersive phenotypes will increase the speed at which species invade new environments, and probably underlies the responses of many species to both past and future climate change.

Neurogenin2 expression in ventral and dorsal spinal neural tube progenitor cells is regulated by distinct enhancers.

The basic helix-loop-helix transcription factor Neurogenin2 (NGN2) is expressed in distinct populations of neural progenitor cells within the developing central and peripheral nervous systems. Transgenic mice containing ngn2/lacZ reporter constructs were used to study the regulation of ngn2 in the developing spinal cord. ngn2/lacZ transgenic embryos containing sequence found 5' or 3' to the ngn2 coding region express lacZ in domains that reflect the spatial and temporal expression profile of endogenous ngn2. A 4.4-kb fragment 5' of ngn2 was sufficient to drive lacZ expression in the ventral neural tube, whereas a 1.0-kb fragment located 3' of ngn2 directed expression to both dorsal and ventral domains. Persistent -gal activity revealed that the NGN2 progenitor cells in the dorsal domain give rise to a subset of interneurons that send their axons to the floor plate, and the NGN2 progenitors in the ventral domain give rise to a subset of motor neurons. We identified a discrete element that is required for the activity of the ngn2 enhancer specifically in the ventral neural tube. Thus, separable regulatory elements that direct ngn2 expression to distinct neural progenitor populations have been defined.

A direct interaction between EXT proteins and glycosyltransferases is defective in hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is an autosomal dominant condition in which bony outgrowths occur from the juxtaepiphyseal regions of the long bones. In a few percent of cases these exostoses undergo malignant transformation to chondrosarcomas. HME results from mutations in one of two homologous genes, EXT1 and EXT2. These are members of a new gene family that is conserved from Caenorhabditis elegans to higher vertebrates. In humans this family comprises five genes which are most conserved at their C-termini, but they do not contain any discernible functional motifs and their function(s) is unclear. Indirect evidence suggests that EXT proteins are involved in glycosaminoglycan synthesis, act as tumor suppressors and affect hedgehog signaling. One recent study has also reported that these proteins co-purify with glycosyltransferase (GlcA and GlcNAc transferase) activity and on that basis it has been postulated that they are themselves glycosyl-transferases. We performed two-hybrid screens with a fragment of EXT2 from the region that is most highly conserved in the gene family and identified two interacting proteins: the tumor necrosis factor type 1 associated protein and a novel UDP-GalNAc:poly-peptide N -acetylgalactosaminyltransferase. Significantly, both these interactions were abrogated by a disease-causing EXT mutation, indicating that they are important in the etiology of HME. The EXT2-GalNAc-T5 interaction provides the first direct physical link between EXT proteins and known components of glycosamino-glycan synthesis.

Molecular cloning and mapping of human semaphorin F from the Cri-du-chat candidate interval.

Cri-du-chat is a human contiguous gene deletion syndrome resulting from hemizygous deletions of chromosome 5p. Here we describe the isolation from within this interval of the human Semaphorin F (SEMAF) gene, a member of a family of proteins that has been implicated in axonal pathfinding. The human SEMAF gene covers at least 10% of the deleted region and defines a new class within this large gene family characterized by the presence of seven type 1 thrombospondin repeats. Prominent expression of murine semaphorin F (Semaf) was observed in the mouse brain, consistent with a role for semaphorin F as a signaling molecule that guides axons or migrating neuronal precursors during development. The known functions of semaphorins and the interesting pattern of expression for Semaf suggest that haploinsufficiency for SEMAF may disrupt normal brain development and might lead to some of the features of Cri-du-chat.

Lack of locomotor-cardiac coupling in trotting dogs.

Coupling of locomotor and cardiac cycles has been suggested to facilitate effective arterial delivery and venous return during vigorous exercise. In an attempt to document locomotor-cardiac coupling, we ran five dogs on a motorized treadmill while monitoring heart activity with surface electrocardiogram electrodes and locomotor events with high-speed video and an accelerometer mounted on the dog's back. Analysis of the cardiac and locomotor frequencies revealed that heart rate was usually slightly greater than stride frequency. Hence the timing of the cardiac cycles varied with respect to the phase of the locomotor cycles, and therefore consistent coupling of the locomotor and cardiac cycles was not observed in any of the dogs. However, the period of the cardiac cycle sometimes varied in a rhythmic way that caused brief periods of transient coupling of the locomotor and cardiac cycles in three of the five dogs. These brief periods of coupling (5-20 heartbeats) occurred at approximately the same phase relationship in each of the three dogs. We hypothesize that the variation in cardiac period and the resulting transient coupling are a function of locomotor and ventilatory influences on venous return and/or ventricular ejection. Because venous return and ejection fraction are likely to vary in an unpredictable manner when animals run in a complex environment, we suggest that reflex control of heart rate will be important during locomotion and strict integer coupling of the locomotor and cardiac cycles is unlikely to evolve.

Presenilin 1 interaction in the brain with a novel member of the Armadillo family.

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed delta-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with beta-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Dishevelled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

Presenilin 1 interaction in the brain with a novel member of the Armadillo family.

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed gamma-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with gamma-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Disheveled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

Isolation of cDNAs from the Cri-du-chat critical region by direct screening of a chromosome 5-specific cDNA library.

Chromosome-specific cDNA libraries are new tools for the isolation of genes from specific genomic regions. We have used two YACs than span the approximately 2-Mb cri-du-chat critical region (CDCCR) of chromosome 5p to directly screen a chromosome 5-specific (CH5SP) fetal brain cDNA library. To compare this library with other sources for new gene discovery, the YACs were hybridized to a normalized infant brain (NIB) cDNA library that has been used extensively for expressed sequence tag (EST) generation. These screens yielded 12 cDNAs from the CH5SP fetal brain library and four cDNAs from the NIB library that mapped to discrete intervals within the CDCCR. Four cDNAs mapped within the minimal CDCCR deletion interval, with the remaining cDNAs being located beyond the boundaries. Only one cDNA shared sequence overlap between the CH5SP and NIB sets of clones. None of the remaining 11 CH5SP cDNAs were homologous to EST sequences, suggesting, in common with previous data on these libraries, that chromosome-specific cDNA libraries are a rich source of new expressed sequences. The single cDNA that did overlap with the NIB library contained two copies of a sequence motif shared with thrombospondin, properdin, and several complement proteins. This motif is usually present in adhesive proteins, and appears to mediate cell-cell or cell-substrate interactions. This new thrombospondin-like gene, and the other three cDNAs that map within the CDCCR, represent candidate genes for the cri-du-chat contiguous gene deletion syndrome.

Human chromosome-specific cDNA libraries: new tools for gene identification and genome annotation.

To date, only a small percentage of human genes have been cloned and mapped. To facilitate more rapid gene mapping and disease gene isolation, chromosome 5-specific cDNA libraries have been constructed from five sources. DNA sequencing and regional mapping of 205 unique cDNAs indicates that 25 are from known chromosome 5 genes and 138 are from new chromosome 5 genes (a frequency of 79.5%). Sequence complexity estimates indicate that each library contains -20% of the approximately 5000 genes that are believed to reside on chromosome 5. This study more than doubles the number of genes mapped to chromosome 5 and describes an important new tool for disease gene isolation.

Five novel genes from the cri-du-chat critical region isolated by direct selection.

Cri-du-chat is a well described partial aneusomy resulting from deletion of the short arm of chromosome 5. The hallmark clinical feature of cri-du-chat, a high-pitched monochromatic cry, has recently been localized to 5p15.3, separate from the remaining clinical features of the syndrome, which have been localized to 5p15.2. Five chromosome 5-specific probes from the latter region, designated the cri-du-chat critical region (CDCCR), were used to isolate 30 cosmids from the LANL chromosome 5 specific cosmid library. The 30 framework cosmids were used in a direct selection with three cDNA sources to isolate an initial set of expressed sequences. Nine unique cDNAs were found that hybridized to four discrete sets of cosmids in the CDCCR. The nine cDNAs are novel by sequence database comparisons, and conservatively represent four transcription units. More recently, we have also constructed a YAC contig of the CDCCR which spans approximately 2 Mb. As expected, ESTs derived from the nine novel cDNAs map back to the contig. Limited expression profiles of these cDNAs have been obtained. Two cDNAs that map to one discrete set of cosmids have different expression patterns, suggesting that they represent two different genes and increasing the number of putative genes to five. Further characterization of these genes and the estimated 100 additional genes deleted in cri-du-chat should lead to better diagnostic markers and an understanding of the molecular mechanisms of the disease.

A yeast artificial chromosome contig of the critical region for cri-du-chat syndrome.

Cri-du-chat is a chromosomal deletion syndrome characterized by partial deletion of the short arm of chromosome 5. The clinical symptoms include growth and mental retardation, microcephaly, hypertelorism, epicanthal folds, hypotonia, and a high-pitched monochromatic cry that is usually considered diagnostic for the syndrome. Recently, a correlation between clinical features and the extent of the chromosome 5 deletions has identified two regions of the short arm that appear to be critical for the abnormal development manifested in this syndrome. Loss of a small region in 5p15.2 correlates with all of the clinical features of cri-du-chat with the exception of the cat-like cry, which maps to 5p15.3. Here we report the construction of a YAC contig that spans the chromosomal region in 5p15.2 that plays a major role in the etiology of the cri-du-chat syndrome. YACs that span the 2-Mb cri-du-chat critical region have been identified and characterized. This YAC contig lays the groundwork for the construction of a transcriptional map of this region and the eventual identification of genes involved in the clinical features associated with the cri-du-chat syndrome. It also provides a new diagnostic tool for cri-du-chat in the shape of a YAC clone that may span the entire critical region.

myc family DNA amplification in small cell lung cancer patients' tumors and corresponding cell lines.

Tumor specimens procured from 38 different small cell lung cancer patients were studied for DNA amplification of the myc family of protooncogenes (c-myc, N-myc, and L-myc). Six of the 38 specimens (16%) had 4-fold or greater myc family DNA amplification (N-myc in 4 and L-myc in 2). All 6 tumors with amplification came from patients who had received combination chemotherapy. The myc family gene copy number of the DNA prepared from 9 tumor cell lines established from these 38 patients was similar to the myc family gene copy number in the DNA prepared from fresh tumor specimens from these same patients. myc family DNA amplification is present in 16% of small cell lung cancer patients' tumors and the amplification pattern in the tumor cell lines is representative of the fresh tumors obtained from the same patients.

Dactinomycin treatment of murine lupus erythematosus. I. Renal disease and longevity.

Three groups of female (NZB X NZW)F1 hybrid mice were treated with an intermittent regimen of dactinomycin (actinomycin D), 3.5 microgram. daily. Median survival was doubled in two of the groups and increased by more than 75 per cent in the third. Most of the treated animals never had significant proteinuria. When kidneys from 14 treated mice, which died between the ages of 11 and 20 months, were examined by light and fluorescence microscopy, most showed the lesions of normal aged CBA and C57BL/6 mice, some expansion of the mesangial matrix and increased cellularity, consistent with deposition of immunoglobulins and complement components in the mesangium, generally sparing the capillary loops. Four of the 14 animals, three of them long-lived, had advanced renal glomerular disease. These data indicate that dactinomycin, by whatever therapeutic mechanism, permits very extended survival of B/W female mice, the large majority of them without significant renal disease.

C4 in glomerular lesions of NZB/NZW mice.

Antisera to human C4 can discriminate circulating Ss protein (C4) levels in mice. Since there has been no information on early complement components (C1, C4, C2) in the renal lesions of B/W mice, we applied the indirect immunofluorescence technique to post-mortem sections of kidney from B/W female mice with advanced renal disease. C4 was present in fifteen of the sixteen specimens, usually in a distribution similar to that of IgG or C3. Specificity was demonstrated by differential absorptions with high-Ss serum from C57BL/6 male mice and low-Ss serum from C3H/HeJ female mice. High-Ss-absorbed antiserum did not stain, while low-Ss-absorbed antibody retained much of its activity. This finding parallels the demonstration of early complement components in lesions of clinical lupus nephritis, and is consistent with classic complement pathway activation in B/W disease.