Xenopus nucleosome assembly protein becomes tissue-restricted during development and can alter the expression of specific genes.

Nucleosome assembly proteins have been identified in all eukaryotic species investigated to date and their suggested roles include histone shuttle, histone acceptor during transcriptional chromatin remodelling and cell cycle regulator. To examine the role of these proteins during early development we have isolated the cDNA encoding Xenopus NAP1L, raised an antibody against recombinant xNAP1L and examined the expression pattern of this mRNA and protein. Expression in adults is predominantly in ovaries. This maternal protein remains a major component of xNAP1L within the embryo until swimming tadpole stages. xNAP1L mRNA is initially throughout the embryo but by gastrula stages it is predominantly in the presumptive ectoderm. Later, mRNA is detected in the neural crest, neural tube, eyes, tailbud and ventral blood islands. In order to test whether xNAP1L has a potential role in gene regulation we overexpressed this protein in animal pole explants and tested the effect on expression of a series of potential target genes. The mRNA encoding the transcription factor GATA-2 was markedly up-regulated by this overexpression. These data support a role for xNAP1L in tissue-restricted gene regulation.

Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells.

Based on evidence suggesting similarities to human embryonic stem cells, human embryonic germ (hEG) cells have been advocated as an alternative pluripotent stem cell resource but have so far received limited attention. To redress this imbalance, human fetal gonads were collected for the isolation and culture of primordial germ cells at 7-9 weeks postconception. We provide evidence for the derivation, culture, and differentiation of hEG cells in vitro. This evidence includes the expression of markers characteristic of pluripotent cells, the retention of normal XX or XY karyotypes, and the demonstration of pluripotency, as suggested by the expression of markers indicative of differentiation along the three germ lineages (ectoderm, mesoderm, and endoderm) and an associated loss of pluripotent markers. In assessing this differentiation, however, we also demonstrate a hitherto unacknowledged overlap in gene expression profiles between undifferentiated and differentiated cell types, highlighting the difficulty in ascribing cell lineage by gene expression analyses. Furthermore, we draw attention to the problems inherent in the management of these cells in prolonged culture, chiefly the difficulty in preventing spontaneous differentiation, which hinders the isolation of pure, undifferentiated clonal lines. While these data advocate the pursuit of pluripotent hEG cell studies with relevance to early human embryonic development, culture limitations carry implications for their potential applicability to ambitious cell replacement therapies.

Mutation of ALMS1, a large gene with a tandem repeat encoding 47 amino acids, causes Alström syndrome.

Alström syndrome (OMIM 203800) is an autosomal recessive disease, characterized by cone-rod retinal dystrophy, cardiomyopathy and type 2 diabetes mellitus, that has been mapped to chromosome 2p13 (refs 1-5). We have studied an individual with Alström syndrome carrying a familial balanced reciprocal chromosome translocation (46, XY,t(2;11)(p13;q21)mat) involving the previously implicated critical region. We postulated that this individual was a compound heterozygote, carrying one copy of a gene disrupted by the translocation and the other copy disrupted by an intragenic mutation. We mapped the 2p13 breakpoint on the maternal allele to a genomic fragment of 1.7 kb which contains exon 4 and the start of exon 5 of a newly discovered gene (ALMS1); we detected a frameshift mutation in the paternal copy of the gene. The 12.9-kb transcript of ALMS1 encodes a protein of 4,169 amino acids whose function is unknown. The protein contains a large tandem-repeat domain comprising 34 imperfect repetitions of 47 amino acids. We have detected six different mutations (two nonsense and four frameshift mutations causing premature stop codons) in seven families, confirming that ALMS1 is the gene underlying Alström syndrome. We believe that ALMS1 is the first human disease gene characterized by autosomal recessive inheritance to be identified as a result of a balanced reciprocal translocation.

Methods for the analysis of protein-chromatin interactions.

The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recognition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecular protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein-DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review techniques used in the investigation of protein interactions with defined nucleosomal templates.